Analysis engine and database for manipulating parameters for fluidic systems on a chip

ABSTRACT

Systems for managing workflows to perform chemical or biological reactions using microfluidic devices.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a division of Ser. No. 11/408,612, filed Apr. 20,2006; which claims priority to U.S. Provisional Application No.60/673,628, filed Apr. 20, 2005. The disclosures of Ser. Nos. 11/408,612and 60/673,628 are hereby incorporated by reference herein for allpurposes.

COPYRIGHT NOTICE

A portion of the disclosure of this patent document contains materialwhich is subject to copyright protection. The copyright owner has noobjection to the facsimile reproduction by anyone of the patent documentor the patent disclosure as it appears in the Patent and TrademarkOffice patent file or records, but otherwise reserves all copyrightrights whatsoever.

BACKGROUND OF THE INVENTION

Over the years, various fluid based processing techniques have beenperformed. Such processing techniques occur in both chemical andbiological arts. Merely as an example, crystallization has been animportant technique to the biological and chemical arts. Specifically, ahigh-quality crystal of a target can be analyzed by x-ray diffractiontechniques to produce an accurate three-dimensional structure of thetarget. This three-dimensional structure information can then beutilized to predict functionality and behavior of the target.

In theory, the crystallization process is often simple to describe. Forexample, a target compound in pure form is dissolved in solvent. Thechemical environment of the dissolved target material is then alteredsuch that the target is less soluble and reverts to the solid phase incrystalline form. Such change in the chemical environment is typicallyaccomplished by introducing a crystallizing agent that makes the targetmaterial less soluble, although changes in temperature and pressure canalso influence solubility of the target material.

In practice however, forming a high quality crystal using conventionaltechniques is generally difficult, or sometimes impossible, requiringmuch trial and error and patience on the part of the researcher. Ahighly complex structure of even simple biological compounds often meansthat they are not amenable to forming a highly ordered crystallinestructure. Therefore, a researcher must often be patient and methodical.The researcher also often experiments with a large number of conditionsfor crystallization, altering parameters such as reagents (e.g., typeand concentration), sample concentration, solvent type, counter solventtype, temperature, and duration in order to grow a high quality crystal,if in fact a crystal can be grown at all. Additionally, conventionaltechniques are often difficult to use and monitor due to long processingtimes often associated with forming detecting, analyzing, crystalstructures.

To overcome certain shortcomings with the conventional techniques,Hansen, et al., describe in PCT publication WO 02/082047, published Oct.17, 2002 and herein incorporated by reference in its entirety for allpurposes and the specific purposes disclosed therein and herein, ahigh-throughput system for screening conditions for crystallization oftarget materials, for example, proteins. The system is provided in amicrofluidic device wherein an array of metering cells is formed by amultilayer elastomeric manufacturing process. Each metering cellcomprises one or more of pairs of opposing chambers, each pair being influid communication with the other through an interconnectingmicrofluidic channel, one chamber containing a protein solution, and theother, opposing chamber, containing a crystallization reagent. Along thechannel, a valve is situated to keep the contents of opposing chambersfrom each other until the valve is opened, thus allowing free interfacediffusion to occur between the opposing chambers through theinterconnecting microfluidic channel. As the opposing chambers approachequilibrium with respect to crystallization and protein concentrationsas free interface diffusion progresses, it is hoped that the proteinwill, at some point, form a crystal. The microfluidic devices taught byHansen et al. are have arrays of metering cells containing chambers forconducting protein crystallization experiments therein. Use of sucharrays in turn provides for high-throughput testing of numerousconditions for protein crystallization which require analysis.

From the above, it is seen that improved techniques for processing andoperating microfluidic chips are highly desired.

SUMMARY OF THE INVENTION

The present invention relates generally to systems and methods formanaging workflow related to processing of one or more microfluidicdevices. Microfluidic devices include a microfluidic chip or device.More particularly, the invention provides a system for automatedpreparation, processing, imaging, analysis, and control of microfluidicdevices used to perform biological and chemical reactions including, forexample, protein crystallization, polynucleotide amplificationreactions, immunological reactions, chemical synthesis, genotyping, andthe like. Merely by way of example, the techniques for microfluidicsystems are applied to protein crystallization experiments using theTOPAZ™ BIOMARK™ and MATRIX™ systems of Fluidigm Corporation of South SanFrancisco, Calif., but it would be recognized that the invention has amuch broader range of applicability. Examples of certain microfluidicdevices and related systems and methods can be found in InternationalPublication Numbers WO 03/085379 A3, WO 2004/089810 A2, PCT/US04/40864,and U.S. patent application Ser. No. 11/006,522, commonly assigned, andhereby incorporated by reference for all purposes.

In a specific embodiment of the present invention, a system for managingworkflow related to processing of one or more microfluidic devices isprovided. The system includes a processor device, a first databasecoupled to the processor device, and one or more process designsprovided within a portion of the first database. The one or more processdesigns are associated with a respective process. The system alsoincludes a microfluidic device comprising one or more well regions. Eachof the well regions is capable of processing one or more of the processdesigns associated with the one or more respective processes. The systemfurther includes an image acquisition device coupled to the processordevice. The image acquisition device is spatially disposed to capture atleast one image of a portion of at least one of the well regions of themicrofluidic device. The image from the image acquisition device is in afirst format and the at least one image of the portion of the at leastone of the well regions may include a portion of an entity. Moreover,the system includes an image processing system coupled to the imageacquisition device for processing the at least one image of the portionof the at least one of the well regions of the microfluidic device inthe first format to a second format and a database management processcoupled to the image processing system.

According to a specific embodiment, the client device is one of aplurality of client devices. In another specific embodiment, the clientdevice is maintained in a private network. Moreover, in an embodiment,the first database and the second database are provided on a singledatabase platform or are provided on multiple database platforms. Inanother embodiment, the well region is coupled to a movable valve memberand the movable valve member is in communication with a driving fluid.In an alternative embodiment, the client device is selected from apersonal computer, a paging device, a cellular phone, a laptop computer,a work station, or other remote computing entity. The system may alsoinclude an experimental manager process coupled to the one or moreprocess designs. In a particular embodiment, the client device includesa user interface device. In another particular embodiment, the one ormore process designs are experimental designs, for example, proteincrystallization experimental designs.

The system also has an image acquisition device (e.g., CCD camera, CIDcamera, CMOS arrays, photographic devices) coupled to the processordevice. The image acquisition device is spatially disposed to capture atleast one image of a portion of at least one of the well regions of themicrofluidic device. The image from the image acquisition device is in afirst format, e.g., pixel domain. The image of the portion of the atleast one of the well regions may include a portion of an entity, e.g.,protein, polynucleotide (e.g., DNA or RNA), cell, chemical, livingtissue. The system has an image processing system coupled to the imageacquisition device for processing the at least one image of the portionof the at least one of the well regions of the microfluidic device inthe first format to a second format, e.g., transform domain. The systemalso includes a database management process coupled to the imageprocessing system and a second database coupled to the databasemanagement system. In a preferred embodiment, the second database isadapted to store an electronic representation of at least the one imagein the second format. A publisher process is coupled to the seconddatabase. The publisher process is adapted to format informationassociated with the electronic representation of the one image in thesecond format. The publisher process is coupled to a world wide areanetwork of computers, e.g., Internet. The system also has a clientdevice (e.g., computer, work station, cell phone, laptop computer, PDA,pager) coupled to the publisher process through at least the world widenetwork of computers. The client device is adapted to allow the user ofthe one or more process designs to provide feedback to at least thedecision making process of the one or more process designs.

In another specific embodiment, a database system for processing imagesprovided in one or more well regions of a microfluidic device isprovided. Each of the one or more well regions are arranged in a spatialorientation. The database system also includes an image capturing devicecoupled to the microfluidic device. The image capturing device isadapted to capture a plurality of images from at least one of the one ormore well regions. Each of the plurality of images is captured in afirst format. The database system additionally includes an imageprocessing device operably coupled to the image capturing device toprovide the plurality of images to the image processing device. Theimage processing device is adapted to process at least a first image anda second image derived from the plurality of images, the first image andthe second image being in a second format, to determine at least a firstfeature information and a second feature information from the respectivefirst image and the second image. The database system further includes adatabase storage device comprising a database management element. Thedatabase storage device is coupled to the image processing device andthe database storage device is adapted to store at least the firstfeature information and the second feature information.

According to a particular embodiment, each of the one or more wellregions are capable of holding a plurality of protein crystals to beimaged. In another embodiment, the first feature information includes afirst fluorescent or chemiluminescent signal and the second featureinformation includes a second fluorescent or chemiluminescent signal.Moreover, in yet another embodiment, the first format and the secondformat are the same format. In a specific embodiment, the first formatand the second format are a pixel format. In another specificembodiment, the first format is a pixel format and the second format isa transform format.

In yet another specific embodiment of the present invention, a computerprogram product for populating one or more databases with informationrelated to an effect of a project is provide. In this embodiment, atleast one structure associated with an entity is provided in a wellregion, the well region selected from a plurality of well regions in aspatial orientation. The computer program product includes code forreceiving, from a user, a reagent and sample information related to theproject. The computer program product additionally includes code forreceiving, from the plurality of well regions, a well region at a time,one or more images of a plurality of objects resulting from aninteraction involving a reagent. The computer program product furtherincludes code for determining from the images of the plurality ofobjects a plurality of features of the plurality of objects. Thecomputer program product further also includes code for populating adatabase with the plurality of features. Moreover, the computer programproducts includes code for publishing the plurality of features to theuser, code for the user to modify the reagent and sample information,and a computer readable storage medium for holding the codes.

In an embodiment, the computer readable storage medium is provided inone or more locations. Additionally, in another embodiment, the codesare provided in one or more locations on the computer readable storagemedium. In another embodiment, the codes are characterized as anexecutable computer code or codes. Furthermore, in a particularembodiment, the code for publishing is provided on a server.

In an alternative embodiment of the present invention, a system forperforming one or more microfluidic processes is provided. The systemincludes an integrated fluidic device comprising a plurality of wellregions and a plurality of control valves. At least one of the controlvalves is coupled to at least one of the well regions. The system alsoincludes a workflow manager and a transfer robot adapted to transfer theintegrated fluidic device between a plurality of stations in response toa series of instructions from the workflow manager. The system furtherincludes a first station comprising a dispensing robot adapted todispense at least one of a plurality of sample solutions and at leastone of a plurality of reagents into the integrated fluidic device. Thesystem additionally includes a second station including a fluidiccontroller unit adapted to actuate at least one of the plurality ofcontrol valves. Moreover, the system includes a third station comprisingan inspection station adapted to acquire and process an image of atleast a portion of one sample well selected from the plurality of samplewells of the integrated fluidic device. In some alternative embodiments,the system further includes a fourth station comprising an integratedfluidic device hotel.

In an embodiment, the one or more crystallization processes comprises anexperiment. In another embodiment, the transfer robot is a track robot.Moreover, in a specific embodiment, the transfer robot includes anarticulated arm.

In another alternative embodiment of the present invention, a method ofdesigning a protein crystallization process is provided. The methodincludes selecting a sample to be screened during a screening process.According to the present invention, the sample is screened for proteincrystallization activity. The method also includes selecting a pluralityof screening reagents utilized during the screening process andselecting a particular type of microfluidic device from a plurality ofmicrofluidic device types.

In a specific alternative embodiment, the method also includes selectinga dispense scheme that includes a mapping from a screening reagent plateto the selected particular type of microfluidic chip. The methodadditionally includes selecting a workflow template from a plurality ofworkflow templates, the workflow template including a work ordersequence and a timing sequence for the protein crystallization process,selecting an owner for the protein crystallization process. The methodfurther includes saving an electronic representation of informationrelated to the previous steps of selecting in a design database.Moreover, the method includes notifying the owner of the proteincrystallization process of a readiness for performing the proteincrystallization process. In a particular embodiment, the method includesperforming the protein crystallization process.

In some embodiments, the method includes entering sample informationinto an experiment manager database. In other embodiments, the sampleinformation includes sample tracking information including name, source,and barcode identifier, protein construct and concentration,co-crystallization components and concentrations, and sample buffercomponents and concentrations. In another embodiment, the methodincludes entering screening reagent information into the experimentmanager database. Additionally, in an embodiment, the screening reagentinformation includes reagent tracking information including reagentname, a source, type of microfluidic device, and reagent barcodeidentifier, expected reagent formation including salt, precipitant, andbuffer. In a particular embodiment, the microfluidic device is a type ofTOPAZ™ Chip. In another particular embodiment, the proteincrystallization process is a protein crystallization experiment. In anembodiment, the method also includes entering a workflow template intothe experiment manager database. In a specific embodiment, the workflowtemplate includes at least one characteristic selected from a chiphydration level, a microfluidic device protocol including pressure leveland loading time at which a reagent and a sample are loaded into themicrofluidic device, an active free interface diffusion protocolincluding a time period during which an interface line is open, a flagrelated to acquisition of a time t₀ image, a number of images to beacquired, and a timing sequence for acquisition of the number of images.Moreover, in some embodiments, the owner of the protein crystallizationprocess is a technician. In another embodiment, notifying isaccomplished via e-mail, text message, voice mail, telephoniccommunication, and/or pager activation.

In yet another alternative embodiment of the present invention, a methodof performing a process utilizing a microfluidic device is provided. Themethod includes preparing the microfluidic device according to apreselected workflow protocol. The method also includes providing atleast one sample tube, a plurality of screening reagent sources, and atleast one microfluidic device at an input stack. The method furtherincludes transferring the at least one sample tube, the plurality ofscreening reagent sources, and the at least one microfluidic device to adispensing robot. The method also includes dispensing at least onesample into the at least one microfluidic device and dispensing at leastone screening reagent into the at least one microfluidic device. Themethod additionally includes transferring the at least one sample tubeand the plurality of screening reagent sources to an output stack.

The method further includes transferring the at least one microfluidicdevice to a first station. In a particular embodiment, the first stationis a free interface diffusion crystallizer. The first station is adaptedto load the at least one sample and the at least one screening reagentinto the at least one microfluidic device. Moreover, the method includestransferring the microfluidic device to an inspection workstation andacquiring a time t₀ image of the microfluidic device. The methodadditionally includes transferring the microfluidic device to a secondstation to initiate active free interface diffusion. In someembodiments, the second station is also a free interface diffusioncrystallizer. The method also includes transferring the microfluidicdevice to a device hotel, which is a microfluidic device hotel in anembodiment according to the present invention. The method furtherincludes transferring the microfluidic device to a third station (e.g.,a free interface diffusion crystallizer) to terminate the active freeinterface diffusion process. In a specific embodiment, the firststation, the second station, and the third station are free interfacediffusion crystallizers.

In some embodiments, the crystallization parameter is a crystal ranking.Moreover, in other embodiments, the method further includes receiving aninput from the user in response through the user interface.

Furthermore, the method includes transferring the microfluidic device toan inspection workstation. The method additionally includes acquiring animage of the microfluidic device at a second time and processing theimage of the microfluidic device acquired at the second time todetermine a crystallization parameter. The method further includesupdating a database to include the crystallization parameter, notifyinga user regarding the updating of the database, and providing a userinterface to communicate the crystallization parameter to the user.

According to yet another alternative embodiment according to the presentinvention, a method of optimizing a protein crystallization experimentis provided. The method includes performing a first screening experimentrelated to protein crystallization using a first reagent set andproducing a first set of experimental results. The method also includesdetermining a new reagent set based on the first reagent set and thefirst set of experimental results. The method further includes providinga plurality of stock solution tubes at an input stack. Each tubecontains a particular stock solution. The method also includes providingan empty reagent plate at an input stack. The method additionallyincludes preparing the new reagent set on the reagent plate from theparticular stock solutions contained in the stock solution tubes andtransferring the plurality of stock solution tubes to an output stack.Moreover, the method includes preparing a microfluidic device accordingto a preselected workflow protocol. Furthermore, the method includestransferring the reagent plate from the input stack to a dispensingstation and transferring the microfluidic device to the dispensingstation. The method also includes selecting a dispense protocol thatincludes a mapping from the reagent plate to the microfluidic device anddispensing the new reagent set from the reagent plate to themicrofluidic device utilizing the dispense protocol. Additionally, themethod includes selecting a workflow template from a plurality ofworkflow templates. The workflow template includes a work order sequenceand a timing sequence for the protein crystallization experiment.Further, the method includes selecting an owner for the proteincrystallization experiment and saving an electronic representation ofinformation related to the previous steps of selecting in a designdatabase. The method also includes notifying the owner of the proteincrystallization experiment of a readiness for performing the proteincrystallization experiment and performing the protein crystallizationexperiment.

In a particular embodiment of the present invention, an automated systemfor processing one or more entities is provided. The system includes aplatform and a robot device comprising a robot arm disposed on theplatform. The robot arm is capable of accessing one or more workstations on the platform. Additionally, the robot is adapted to transferone or more microfluidic devices from a first spatial location to asecond spatial location. In this particular embodiment, the one or moremicrofluidic devices include one or more reaction chambers therein. Thesystem additionally includes an input device coupled to the one or morework stations. The input device is adapted to receive one or moremicrofluidic devices from a user. The system also includes an outputdevice coupled to one or more workstations. The output device is adaptedto output the one or more microfluidic device to a user. Moreover, thesystem includes an image capturing workstation coupled between the inputdevice and the output device. The image capturing workstation is adaptedto capture one or more images of a portion of one or more reactionchambers and any contents therein of the microfluidic chip. The systemfurther includes one or more ports coupled to one or more microfluidicdevices. The one or more ports are adapted to provide one or more inputsinto at least one of the reaction chambers. Moreover, the one or moreports are adapted to manipulate one or more processes being carried outin at least one of the reaction chambers in the one or more microfluidicdevices.

In an embodiment, a chip hotel is coupled to the image capture deviceand is adapted to house one or more microfluidic devices therein in apredetermined environment. In a particular embodiment, the chip hotel isadapted to maintain a predetermined level of electromagnetic radiation.Moreover, in another embodiment, the one or more processes include atemporal element or a temperature element. Additionally, in anembodiment, the one or more ports is adapted to provide one or morefluids to the one or more microfluidic devices. In another embodiment,the robot device includes a track that is spatial disposed from a firstspatial region to a second spatial region on the platform. In anembodiment, the system further includes a robot controller coupled tothe robot device. The robot controller includes a plurality ofinput/output ports, each of the input/output ports being adapted toreceive or transfer one or more electrical signals to operate the robotdevice. In some embodiments, the image capturing device includes a CCDdevice. In other embodiments, the system further includes a back endwork station coupled between the input device and the output device.

In another particular embodiment, a graphical user interface device fora fluidic micro chip analysis system is provided. The graphical userinterface device includes a first portion on a display comprising arepresentation of an array of well regions. The array of well regionsincludes a first axis, including a first set of indications. The arrayof well regions also includes a second axis, including a second set ofindications. Each of the well regions in the array are capable of beingaddressed via at least one of the first indications and at least one ofthe second indications. The graphical user interface device alsoincludes a second portion on the display comprising a list of entities.The list of entities are associated with at least one of the wellregions in the array. Moreover, the list of entities are displayed upona selection of the one of the well regions in the array.

In a particular embodiment, the first indications are numbered from 1through N and the second indications are numbered from 1 through M. Inanother embodiment, the user interface also includes a third portion onthe display indicating bar code information. In yet another embodiment,the user interface further includes a fourth portion on the displayindicating an identifier. In some embodiments, the array of well regionsincludes at least one sample in one of the well regions. In otherembodiments, the list of entities includes at least one entity. In aparticular embodiment, the list of entities includes at least a resultfor at least one of the well regions.

In yet another particular embodiment, a method for displaying featureson a graphical user interface device for a fluidic micro chip analysissystem is provided. The method includes outputting on a first portion ofa display a representation of an array of well regions. The array ofwell regions includes a first axis, including a first set ofindications. The array of well regions also includes a second axis,including a second set of indications. Each of the well regions in thearray is capable of being addressed via at least one of the firstindications and at least one of the second indications. The method alsoincludes outputting on a second portion of the display a list ofentities. The list of entities is associated with at least one of thewell regions in the array. Additionally, the list of entities isdisplayed upon a selection of the one of the well regions in the array.

In a specific embodiment, the list of entities includes one or morereagents. Moreover, in another embodiment, the array includes at least96 well regions of a reagent plate. In some embodiments, the firstportion and the second portion are displayed simultaneously. Moreover,in an embodiment, the first indications are numbered from 1 through Nand the second indications are numbered from 1 through M. In anotherembodiment, the method further includes a third portion on the displayindicating bar code information. In a particular embodiment, the methodalso includes a fourth portion on the display indicating an identifier.

According to one embodiment, the array of well regions includes at leastone sample in one of the well regions. Additionally, in an embodiment,the list of entities includes at least one entity. In a particularembodiment, the list of entities includes at least a result for at leastone of the well regions.

In an alternative particular embodiment according to the presentinvention, a method for displaying features on a graphical userinterface device for a fluidic micro chip analysis system is provided.The method includes outputting on a first portion of a display arepresentation of an array of well regions. The array of well regionsincludes a first axis, including a first set of indications, and asecond axis, including a second set of indications. Each of the wellregions in the array is capable of being addressed via at least one ofthe first indications and at least one of the second indications. Themethod also includes outputting on a second portion of the display alist of entities. The list of entities is associated with at least oneof the well regions in the array. The list of entities is displayed upona selection of one of the one of the well regions in the array.

In some embodiments, the first portion, the second portion, and thethird portion are displayed simultaneously. In other embodiments, themicrofluidic chip includes a plurality of well regions and aninput/output region. Moreover, in an embodiment, the list of addressesincludes the first set of indications and the second set of indications.In a particular embodiment, the first set of indications is numberedfrom 1 through N and the second set of indications is numbered from 1through M. Furthermore, in an embodiment, the first portion, the secondportion, and the third portion are displayed concurrently with anoperation process associated with the microfluidic chip. In a specificembodiment, the first portion and the second portion are differentportions on the display. Moreover, in another embodiment, the array ofwell regions is associated with a microtiter plate.

In yet another alternative particular embodiment, a graphical userinterface device for a fluidic micro chip analysis system coupled to adatabase is provided. The graphical user interface device includes afirst portion provided on a display of a representation of an array ofwell regions. The array of well regions includes a first axis, includinga first set of indications. The array of well regions also includes asecond axis, including a second set of indications. Each of the wellregions in the array is capable of being addressed via at least one ofthe first indications and at least one of the second indications. Thegraphical user interface device also includes a second portion providedon the display of a list of addresses. Each of the addresses isrespectively associated with well regions in the array. The graphicaluser interface device further includes a third portion provided on thedisplay of a representation of a microfluidic chip.

In a specific embodiment, the first portion, the second portion, and thethird portion are displayed simultaneously. In another specificembodiment, the microfluidic chip includes a plurality of well regionsand an input/output region. Moreover, in an embodiment, the list ofaddresses includes the first set of indications and the second set ofindications. In another embodiment, the first set of indications isnumbered from 1 through N and the second set of indications is numberedfrom 1 through M. In a particular embodiment, the first portion, thesecond portion, and the third portion are displayed concurrently with anoperation process associated with the microfluidic chip. Additionally insome embodiments, the first portion and the second portion are differentportions on the display. In an embodiment, the array of well regions isassociated with a microtiter plate.

According to a particular embodiment according the present invention, acommunications system is used to transmit information related to aprotein crystallization process to a remote user. The communicationssystem may include, for example, email, voice mail, instant messaging,paging, and/or SMS. In another embodiment, the microfluidic elastomericprocess includes at least one of a protein crystallization process, afluorogenic reaction process, or a chemiluminescent reaction process.

In another particular embodiment, methods and systems according to thepresent invention provide for the use of a database to manageinformation related to processes performed using microfluidicelastomeric chips. In some embodiments, the information is related to aprotein crystallization process. In other embodiments, the informationis related to at least one of a fluorogenic reaction process or achemiluminescent reaction process.

In another alternative embodiment, a method for operating an imagingsystem for biological applications is provided. The method includescapturing one or more images, for example, of a portion of a proteincrystal, in a pixel domain. The pixel domain is associated with a wellregion from a microfluidic chip. Moreover, the image in the pixel domainis captured using an image capturing device. The method also includesprocessing the one or more images in the pixel domain to deriveinformation associated with the one or more images. In a specificembodiment, the information relates to crystallization process. Themethod further includes transferring a portion of the informationassociated with the one or more images to a remote client device. Merelyby way of example, in a particular embodiment, the remote client deviceis a computer, work station, cell phone, laptop computer, PDA, or pager.According to an embodiment of the present invention, transferringoccurs, in part, through a network of computers (e.g., the Internet).

In an embodiment, the information relates to a crystallization processand the one or more images includes a portion of a crystal from thecrystallization process. In another embodiment, the information relatesto at least one of a fluorogenic reaction process or a chemiluminescentreaction process and the one or more images includes images of at leastone of a fluorescent chemical species or a luminescent chemical species.In other embodiments, the information relates to a process performed ina microfluidic elastomeric device. In some embodiments, the methodfurther includes storing the information in a portion of a database, thedatabase being coupled to the image capturing device. In a particularembodiment, the micro fluidic chip includes the well region coupled to avalve region and the valve region is coupled to one of a plurality ofinput and/or output ports.

In another embodiment, the method also includes using the portion ofinformation for a drug discovery process. In a particular embodiment,the processing includes a publication process for the remote clientdevice. In other embodiments, the processing includes converting the oneor more images in the pixel domain to a second format to be transferredthrough the network of computers.

Numerous benefits are achieved using the present invention overconventional techniques. Some embodiments provide automated systemsemploying computer control to reduce the workload and increase thesystem efficiency for protein crystallization experiments. Additionally,the present invention provides an automated and/or semi-automatedtechnique for processing one or more species in a microfluidic chipenvironment. Depending upon the embodiment, remote access may also beachieved in certain methods and systems. In other embodiments, theinvention provides methods and systems that include partially or fullyintegrated computer based techniques including remote access, databasesystems and methods, and imaging techniques. Moreover, the presentmethods and systems may be integrated with other known and/or futuremethods and systems, including the CrystalMation™ product manufacturedby RoboDesign International Incorporated of Carlsbad, Calif., theCrystalTrak database application, also manufactured by RoboDesignInternational Incorporated, the Rhombix® Series manufactured by KendroLaboratory Products, Inc. of Asheville, N.C., the RockMaker manufacturedby Fommulatrix, Inc., of Waltham, Mass., and others. Depending upon theembodiment, one or more of these benefits may exist. These and otherbenefits have been described throughout the present specification andmore particularly below.

Various additional objects, features and advantages of the presentinvention can be more fully appreciated with reference to the detaileddescription and accompanying drawings that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a simplified diagram illustrating a process workflow accordingto an embodiment of the present invention;

FIG. 2 is a simplified diagram illustrating an automated proteincrystallization management system according to an embodiment of thepresent invention;

FIG. 2A is a simplified schematic diagram illustrating an automatedprotein crystallization system according to an embodiment of the presentinvention;

FIG. 2B is a simplified schematic diagram illustrating another automatedprotein crystallization system according to another embodiment of thepresent invention;

FIG. 2C is a simplified schematic diagram illustrating an automatedprotein crystallization experiment and analysis system according to anembodiment of the present invention;

FIG. 2D is a simplified schematic diagram of system software accordingto an embodiment of the present invention;

FIG. 2E is simplified schematic network diagram illustrating a computernetwork according to an embodiment of the present invention;

FIG. 3 is a simplified diagram illustrating a graphical user interfaceaccording to an embodiment of the present invention;

FIG. 4 is a simplified diagram illustrating a reagent graphical userinterface according to an embodiment of the present invention;

FIG. 5 is a simplified diagram illustrating a Screen Plate Creationgraphical user interface according to an embodiment of the presentinvention;

FIGS. 6A-6D are simplified diagrams illustrating additional graphicaluser interfaces according to an embodiment of the present invention;

FIG. 7 is a simplified flowcharts illustrating a method of designing anexperiment according to an embodiment of the present invention;

FIG. 8 is a simplified flowchart illustrating a method of running anexperiment according to an embodiment of the present invention;

FIG. 9 is a simplified flowchart illustrating a method of optimizing anexperiment according to an embodiment of the present invention;

FIG. 10 is a simplified flowchart illustrating a method of translatingan experimental design according to an embodiment of the presentinvention;

FIG. 11 is a simplified flowchart illustrating a method of optimizing atranslation of an experimental design according to an embodiment of thepresent invention;

FIG. 12 is a simplified flowchart illustrating a workflow templateaccording to an embodiment of the present invention;

FIGS. 13-26 are simplified flowcharts illustrating operations performedaccording to exemplary embodiments of the present invention;

FIG. 27 is a schematic representation of an exemplary device with amatrix design of intersecting vertical and horizontal flow channels;

FIG. 28 is a plan view of an exemplary blind channel device;

FIGS. 29A and 29B respectively are a cross-sectional view and aschematic diagram of another hybrid type microfluidic device andrepresents the type of device used to conduct experiments according toembodiments of the present invention;

FIG. 30 is a schematic diagram of the microfluidic device used forexperiments according to another embodiment of the present invention;

FIGS. 31A-31D depict two preferred designs of a partitioningmicrofluidic device in a valve off and valve actuated state;

FIG. 32 is a schematic drawing of one “immunochip” embodiment of thedevice having 24 sample inputs and 24 primary antibody inputs, and 24secondary antibody inputs;

FIG. 33 is a schematic drawing of another embodiment of the devicehaving 96 sample inputs and 24 primary antibody inputs, and 24 secondaryantibody inputs;

FIG. 34 is a schematic drawing showing a magnified section of anembodiment the device;

FIG. 35 is a simplified flowchart illustrating a method of performinggene expression/genotyping experiments according to an embodiment of thepresent invention; and

FIGS. 36-40 are simplified flowcharts illustrating operations performedaccording to exemplary embodiments of the present invention;

FIG. 41A depicts a substrate of a microfluidic device that hasintegrated pressure accumulator wells according to an embodiment of thepresent invention;

FIG. 41B depicts an exploded view of the microfluidic device shown inFIG. 41A, and further including an elastomeric block;

FIG. 41C is an overall view of the microfluidic device shown in FIG.41B;

FIG. 41D is a plan view of the microfluidic device shown in FIG. 41B;

FIG. 41E depicts a plan view of the microfluidic device shown in FIG.41B;

FIG. 41F depicts a bottom plan view of the microfluidic device shown inFIG. 41B;

FIG. 41G depicts a cross-sectional view of the microfluidic device shownin FIG. 41B;

FIG. 42A is a perspective view of a station for actuating a microfluidicdevice according to an embodiment of the present invention;

FIGS. 42B and 42D are perspective and side views, respectively, of thestation of FIG. 42A with the platen in a down position;

FIG. 42C is a side view of the station of FIG. 42A with the platen in anup position;

FIG. 42E depicts a close-up view of the platen of FIG. 42A;

FIG. 42F depicts a cut-away side view of the platen of FIG. 42A;

FIG. 42G is a close-up view of a purge actuator acting on a check valveaccording to an embodiment of the present invention;

FIG. 42H depicts a cut-away view of a platen urged against the upperface of a microfluidic device according to an embodiment of the presentinvention;

FIG. 43A is a simplified overall view of a system according to anembodiment of the present invention;

FIG. 43B is a perspective view of a receiving station in the system ofFIG. 43A; and

FIG. 43C is a rear plan view of fluidic routing within a plate interfaceor platen according to another embodiment of the present invention; and

FIG. 44 is a schematic representation of another exemplary matrix designdevice that utilizes guard channels to reduce sample evaporation.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

The invention provides systems and methods for managing workflow relatedto processing of one or more microfluidic devices, for example a systemfor automated preparation, processing, imaging, analysis, and control ofmicrofluidic devices.

FIG. 1 is a simplified diagram illustrating a process workflow 100according to an embodiment of the present invention. This diagram ismerely an example of a process workflow, which should not unduly limitthe scope of the claims herein. One of ordinary skill in the art wouldrecognize many variations, modifications, and alternatives. Asillustrated, the workflow process 100 is generally initiated by enteringreagent and protein sample information in step 110. In some embodiments,the reagent and protein sample information if entered by a system user,although this is not required by the present invention. Additionally,experimental design information (step 112) and design of various chipruns (step 114) is performed according to embodiments of the presentinvention. As discussed more fully below with relation to FIG. 2 and thecorresponding section of the detailed description, the experimentaldesign information along with information regarding the designs for thevarious process runs are generally stored in an accessible database.Generally, a database is defined as one or more large structured sets ofpersistent data, usually associated with software to update and querythe data. A simple database might be a single file containing manyrecords, each of which contains the same set of fields where each fieldis a certain fixed width. A database is one component of a databasemanagement system.

In some embodiments of the present invention, various proteincrystallization processes are referred to as protein crystallizationexperiments. This language is not intended to limit the scope of theclaims herein, but to merely exemplify an application of the methods andsystems provided according to embodiments of the present invention.

In step 116, the microfluidic device selected in step 114 is provided.Additionally, the protein sample and the reagent defined in step 114 areprovided and dispensed into the microfluidic device or chip. In someembodiments, a screening chip is provided, the screening chip adaptedfor running a protein crystallization screening experiment. In otherembodiments, a diffraction chip is provided, the diffraction chipadapted for running a protein crystallization diffraction experiment.Moreover, in yet other embodiments, other chips suitable for performingclassic crystallization methods, for example, microtiter plates areprovided and the protein sample and reagents are dispensed into suchsuitable chips. Additional details regarding the various microfluidicdevices utilized in embodiments of the present invention will beprovided throughout the present specification and more particularlybelow. In some embodiments, the dispensing step 116 includes dispensingof reagent solutions from stock reagent solutions into a reagent platein order to prepare particular reagent solutions. However, as one ofskill in the art will appreciate, this step is not required by thepresent invention.

Process 120 includes a number of individual steps related to certainmicrofluidic chips. In a particular embodiment of the present invention,the microfluidic chips are TOPAZ™ screening and/or TOPAZ™ diffractionchips or devices. Of course, other types of chips may also be useddepending upon the specific embodiment. In step 122, the desired proteinsamples and reagent solutions are loaded into the selected microfluidicdevice and the device is placed in a free interface diffusion (FID)system. Generally, a FID system, such as the TOPAZ™ FID crystallizer(FIDX™), available from the present assignee, is utilized to intermixthe protein sample and the reagents previously loaded into themicrofluidic device, thereby producing crystals depending on theconditions. In a specific embodiment of the present invention, the FIDXis utilized at a screening stage of the process 100, during which anumber of protein samples and reagents are combined in a screeningprocess.

Image acquisition (IA) and image processing (IP) are performed in step128. In some embodiments, these processes are performed using anautomated image acquisition and processing system such as the TOPAZ™AUTOINSPEX™ Workstation (AIX), available from the present assignee. Insome embodiments, the image acquisition system includes an capturingdevice that is capable of capturing a single image of all reactionchambers concurrently or simultaneously. Thus, in contrast with step andrepeat or stitching systems, images of an entire microfluidic device arecollected at the same time, rather than in a sequential manner.Additional details regarding such systems are provided in “Method andSystem for Microfluidic Device and Imaging Thereof,” published asInternational Patent Application No. WO2004/103563A2 filed May 20, 2004and “Image Processing Method and System for Microfluidic Devices,”listed under U.S. patent application Ser. No. 10/902,494, filed Jul. 28,2004, commonly assigned, and hereby incorporated by reference for allpurposes.

A system user is able to view and annotate experimental results in step126 using software. In a specific embodiment of the present invention,software such as the TOPAZ™ AIX Software suite, including Crystal Visionsoftware is used to view the experimental results and further annotatethe experiments as desired. As illustrated by feedback look 128, afterreview of the experimental results, steps 124 and 126 may be repeated asdesired. In some embodiments, a system user determines that additionalimages, for example, should be acquired and analyzed. Accordingly,feedback loop 128 is utilized to accomplish these objectives.

Upon the completion of the steps provided within process 120, feedbackloop 140 is utilized in some embodiments to optimize the screeningprocess. Experimental results produced in process 120 are utilized toredesign the experimental conditions in step 112, redesign the chip runsin step 124, and reinitiate the screening process. In alternativeembodiments, only a portion of the experimental design or a portion ofthe chip run design, or both, are modified, depending on the particularapplication. Feedback loop 140 may be used once, twice, or more times,depending on the particular application and the results obtained duringthe screening runs.

In some embodiments, the screening process, generally combined with thescreening optimization process, results in the production of crystalssuitable for x-ray crystallography in step 146. As is well known to oneof skill in the art, exposure of a crystal structure to a beam ofx-rays, or other particles, may result in the production of diffractionpatterns which are analyzed to determine the structure (step 148) of theparticular crystal structure under examination. Accordingly, forscreening processes which produce crystals amenable to x-ray or otherdiffraction studies, these studies may be performed (line 144), therebygenerating information related to the crystal structure in step 148,completing the process 100.

Not only does process 120 provide for protein crystallization screeningexperiments, it additionally provides for protein crystallizationdiffraction experiments. Merely by way of example, crystals suitable fordiffraction analysis are produced using TOPAZ™ diffraction microfluidicdevices in an embodiment of the present invention. Typically, after thescreening optimization process is performed, the experimental and chiprun designs are modified at steps 112 and 114 and process 120 isrepeated using diffraction chips. In a particular embodiment, aftercrystals have been obtained in the screening loop, the conditions aretranslated from nano-scale FID to micro-scale vapor diffusiontechniques. Merely by way of example, large, diffraction-qualitycrystals are produced in a diffraction phase of process 120 using TOPAZ™diffraction microfluidic devices using conditions determined during thetranslation process.

As discussed in relation to the screening process, feedback loop 128 isprovided within process 120 for iteration on the image scanning andanalysis process. Moreover, feedback loop 142 is provided foroptimization of the diffraction chip experiments. Iteration in thelarger feedback loop 142 provides for modification of design parameters,preferably leading to the production of diffraction-quality crystals. Asdiscussed before, upon production of acceptable crystals, x-raydiffraction experiments (step 146) are utilized in a particularembodiment to generate structure information (step 148), terminating theworkflow process 100.

Embodiments of the present invention are not limited to the productionof diffraction-quality crystals through the use of TOPAZ™ diffractionchips. In alternative embodiments, process 130, including classiccrystallization methods, such as vapor diffusion, are utilized toproduce diffraction-quality crystals. In the vapor diffusion technique,a drop of water containing, for example, a protein sample, stabilizingbuffers, precipitants, and/or crystallization reagents is allowed toequilibrate in a closed system containing a reservoir. The reservoirtypically contains the same ingredients as the drop with the exceptionof the protein. The concentration of the materials in the reservoir istypically higher than the concentration in the drop so that waterpreferentially evaporates from the drop. Given the appropriateconditions, the evaporation will produce a gradual increase in proteinand precipitant concentrations, resulting in the formation of proteincrystals. Other crystal formation processes in addition to vapordiffusion are included in process 130 according to embodiments of thepresent invention.

Image scanning in step 132 and user annotation in step 134 are generallycombined with feedback loops 135 and 136 in processes utilizing thesecrystallization methods. As illustrated in FIG. 1, feedback loop 142 isavailable for optimization of the crystallization methods illustrated byprocess 130. As discussed before, upon production of acceptablecrystals, x-ray diffraction experiments (step 146) are utilized in aparticular embodiment to generate structure information (step 148),terminating the workflow process 100. Although the above systems andmethods for managing workflow have been described generally in relationto certain microfluidic devices such as, for example TOPAZ™, others canalso be used. Other microfluidic devices that can be used in conjunctionwith the invention include devices designed to perform reactionsincluding, but not limited to, polynucleotide hybridizations, PCR(polymerase chain reactions), immunological reactions such as ELISAreactions, signal amplifications, such as those called the INVADER™system manufactured by Third Wave Technologies of 502 Rosa Rd., Madison,Wis. 5371-1256, digital amplifications, including, but not limited to,digital PCR, cell, tissue, microbe assays, crystal formation ofnon-organic materials, protein capture assays, including, quantitativeand/or qualitative protein assays, cell and/or particle sorting,molecular sizing and/or sequencing, and the like. Of course, one ofordinary skill in the art would recognize many variations, alternatives,and modifications. An example of a system for automated proteincrystallization using such a microfluidic chip can be found in moredetail below.

FIG. 2 is a simplified diagram illustrating an automated proteincrystallization management system 200 according to an embodiment of thepresent invention. As illustrated in the figure, the various componentsutilized to perform steps of the workflow discussed in relation to FIG.1 are provided. In some embodiments, the various software componentsprovided in FIG. 2 are part of a TOPAZ™ Database Application Suite,although this is not required by the present invention. For example, anexperiment manager 205 is provided that is utilized to enter, store,retrieve, modify, and process information provided as part of thereagent and sample information entry step 210, the design of theexperiment in step 212, and the design of a chip run in step 214,reagent design, and data mining.

In a particular embodiment, the experiment manager is TOPAZ™ ExperimentManager software, available from the present assignee. In someembodiments, the experiment manager is contained in a first database,although this is not required by the present invention. In alternativeembodiments, the experiment manager is provided as part of the database250, discussed more fully below. In a particular embodiment, theexperiment manager is the main graphical user interface (GUI) for theDatabase Application. Furthermore, the experiment manager interacts withthe database 250 directly through data bus 259. In a specificembodiment, the database 250 is a Microsoft SQL or an MSDE server.

In a specific embodiment of the present invention as illustrated in FIG.2, a number of applications are based on certain enhancements made toproducts available from the present assignee. For example, the FIDcrystallizer 221, the image acquisition and image processing system 223,and the Crystal Vision package 225 are coupled to perform proteincrystallization experiments and exchange information with database 250through server 252 as discussed in more detail below.

In step 216, the protein sample and reagent are dispensed into amicrofluidic chip as discussed previously in preparation for a proteincrystallization experiment. As illustrated in FIG. 2, the softwareincluded in various system components is shown. For example, softwareresident in the FID crystallizer 221, e.g., a TOPAZ™ FIDX, is utilizedto perform the steps of loading a TOPAZ™ chip into the FIDX, openingappropriate valves in the TOPAZ™ chip to initiate FID, and closingappropriate valves as necessary to terminate the FID process (222). Inlike manner, software resident in the image acquisition and processingsystem 223 is adapted to automatically acquire and process images (step224) of at least a portion of a microfluidic device. Using CrystalVision software (225), for example, a user is able to annotate theexperimental results in step 226. In a particular embodiment of thepresent invention, separate TOPAZ™ Image Acquisition and TOPAZ™ AutoProcessor applications are provided as part of application 223.

Data bus 253 couples the FID crystallizer 221, the image acquisition andprocessing system 223, and the Crystal Vision software 225 to a server252. In the embodiment illustrated in FIG. 2, the server is a DatabaseApplication Server (e.g., a TOPAZ™ Data Application Server, sometimesreferred to as a TOPAZ™ Server), but this is not required by the presentinvention. In some embodiments, the server 252 is a hardware device,while in other embodiments, the server 252 is a database managementprocess. For example, in an embodiment, the server is an interface layerbetween the database 250 and various software components, e.g., TOPAZ™software components. In general, the database management process is abackground application. One of ordinary skill in the art would recognizemany variations, modifications, and alternatives. As illustrated in FIG.2, the server 252 is coupled to database 250 by an additional data bus257, although in an alternative embodiment, the server 252 and thedatabase 250 are both coupled to data bus 253. Furthermore, database 250is coupled to the experiment manager 205 through data bus 259, providingfor two way communication between the database management process 252and the experimental design phase of the protein crystallizationprocess.

Data bus 253 provides for two way communication between the databasemanagement process 252 and the software driven processes utilized in theprotein crystallization experiments. Accordingly, data utilized by theapparatus can be provided by the server or other pieces of equipment.Moreover, results and analysis produced by the apparatus are availableto the system user through the server. Furthermore, commands to controlfacets of the protein crystallization processes may be generated at theserver or other location and communicated to the apparatus through thedata bus.

Merely by way of example, image acquisition and processing feedback loop228 is utilized in a specific embodiment through communication over bus253. After a first image acquisition and processing process isperformed, the data is provided to the server 252 and the Crystal Visionsoftware 225. A system user accesses the data through server 252 anddetermines that an additional image scan is appropriate. Communicatingover bus 253, an instruction is provided to system 223 to acquire andprocess a second image, effectuating feedback through loop 228. This ismerely a single example and one of ordinary skill in the art wouldrecognize many variations, modifications, and alternatives.

As discussed in relation to FIG. 1, screening optimization is performedusing feedback loop 240 and diffraction optimization is performed usingfeedback loop 242 in embodiments of the present invention. Moreover,database management process 252 and database 250 are utilized in someembodiments to track the iterations performed along these feedbackloops.

Alternative crystallization methods, such as vapor diffusion, areutilized to produce diffraction-quality crystals as illustrated in FIG.2. For example, software resident on a user's microscope 231 and crystalscoring programs interact with the microscope to collect and analyzeimages (step 232) of a protein crystal in an embodiment of the presentinvention. A user may record annotations related to the proteincrystallization process in step 234 using the score card software 233 asillustrated. As with the information related to the screening anddiffraction processes for microfluidic chips, data bus 253 provides fortwo way communication with the apparatus utilized in these alternativecrystallization methods. Additionally, feedback loops 235 and 236 areprovided as discussed in relation to FIG. 1. Diffraction-qualitycrystals, produced by screening or diffraction methods are subjected toanalysis by system/method 246 and structure information is preferablyascertained at step 248.

In an embodiment of the present invention, the Score Card 233 is asoftware application adapted for a system user to manually enter theexperimental conditions of the microfluidic device (chip) or of aclassic crystallization method. As illustrated, the score card 233interacts with the database 250 through the server 252. In a specificembodiment, the server 252 is a TOPAZ™ Database Application Server.

Not only is the database 250 coupled to the database management process252 in FIG. 2, but it is additionally coupled to publisher 254. In aspecific embodiment of the present invention, the publisher is utilizedto extract data related to the protein crystallization process andtransmit this data to a number of users 270, 272, 274, and 276 throughcomputers 260, 262, 264, and 266. As illustrated in FIG. 2, thecomputers are coupled to internet server 256 through data links 261,263, 265, and 267. In a particular embodiment of the present invention,the publisher is an application that generates experimental results asHTML files and publishes (e.g., copies) them to Internet Server 256.Thus, utilizing embodiments of the present invention, data resultingfrom the protein crystallization process is available for viewing by awider audience without providing direct connections of the audience tothe database 250.

Feedback loop 280 provides a link for users 270-276 to remotely interactwith the Experiment Manager 205. Accordingly, embodiments of the presentinvention provide for automated control of protein crystallizationexperiments, remote publishing of the resulting experimental data andfeedback based control of subsequent protein crystallizationexperiments. As illustrated, the users are provided with access to theDatabase as well as the Server, enabling remote analysis of experimentaldata and subsequent feedback. Merely by way of example, a user couldview an experimental result in Crystal Vision, decide an additionalimage was beneficial, and instruct the AIX to perform an additionalcycle. One of ordinary skill in the art would recognize many variations,modifications, and alternatives. Thus, through embodiments of thepresent invention, users are enabled to interact with, analyze, andcontrol the protein crystallization experimental process at numerousstages of such processes.

In some embodiments of the present invention, a number of controlfeatures are within the control of the Database Application Suite. Anexample of such a system is discussed below in relation to FIG. 2. Inparticular embodiments, the sample and reagent dispensing function isprovided separately from the Database Application Suite. In some ofthese particular embodiments, a user will follow the follow thedispensing instruction provided by the Experiment Manager in order toperform the sample and reagent dispensing functions. In otherembodiments, the sample and reagent dispensing function is provided as afunction under the control of the automated system, as illustrated inFIG. 9 below.

FIG. 2A is a simplified schematic illustration of an automated systemfor performing protein crystallization experiments according to anembodiment of the present invention. As illustrated in FIG. 2A, anautomation table 2050 is provided to provide a stable base for a numberof system components. In some embodiments according to the presentinvention, the entire automation table 2050 and all the items associatedwith the automated system are enclosed in an environmental enclosure,that provides, for example, temperature and humidity control for theautomated system components. In alternative embodiments, partial controlis provided by environmental enclosures that contain a portion of thesystem components that do not provide independent environmental control,for example, the track robot.

Track robot 2052 with articulated arm 2054 is located on the automationtable 2050 and is adapted to provide automated transport operationsbetween other system components on the automation table. Although FIG.2A illustrates robot 2052 as a track robot, this is not required by thepresent invention. Other robots, including articulated robots orcombination track/articulating robots, such as the CRS F3t Track RobotSystem, available from the Thermo Electron Corporation of Waltham, Mass.

In the embodiment illustrated in FIG. 2A, the automated system alsocomprises a number of input/output stacks adapted to store microfluidicdevices or chips. In the embodiment illustrated in FIG. 2A, a UserInput/Output Stack 2056 is provided. The User input/output stack isgenerally utilized for a user to transfer microfluidic chips to and fromthe Microfluidic Chip Automation system. For example, after reagent andsamples have been loaded into microfluidic chips, the chips are placedin the user input/output stack by a robot, system operator, technician,or the like. Typically, a user is a technician, a system operator, andthe like. Moreover, in one embodiment, two automation input/outputstacks 2058 and 2060 are provided at two sides of the automation table.Generally, the Automation Input/Output stacks are used for transferringmicrofluidic chips between different portions of the automation systemor between different automation systems (e.g., systems on differentautomation tables). Of course, one of skill in the art will appreciatethat different numbers and arrangements of the input/output stacks maybe used depending on the particular application.

FIG. 2A also illustrates a microfluidic Chip Hotel 2062 on theautomation table. In some embodiments, the hotel is utilized to storechips before, during, and/or after FID experiments. Merely by way ofexample, the hotel stores chips in between analysis sessions using theAIX. Although FIG. 2A illustrates a microfluidic Chip Hotel, the presentinvention is not limited to chip hotels for microfluidic devices. Inalternative embodiments, the hotel provides a controlled environment forstorage of microtiter plates, trays, and the like.

A microfluidic chip FID Crystallizer (FIDX) 2064, a thermal transferentity 2059, and a microfluidic chip AUTOINSPEX™ Workstation (AIX) 2066are also provided as illustrated in FIG. 2A. As described previously,these system components are typically utilized in performing proteincrystallization experiments.

In an embodiment of the present invention, the automated system isadapted to transfer a microfluidic chip 2068 between the userinput/output stack, the automation input/output stacks, the MicrofluidicChip FIDX, the Microfluidic Chip AIX, and the Microfluidic Chip Hotel. Acomputer (not shown) is utilized to automate and control the operationof the system. In a specific embodiment of the present invention, thesystem is a protein crystallization experiment system. Merely by way ofexample, under computer control and through the use of the softwaredescribed herein, a microfluidic chip may be transferred from one of theinput/output stacks, processed through the FIDX, placed in the chiphotel for a predetermined period, and transferred to the AIX at apredetermined time. After automated processing, experimental results,communicated through the software and systems described herein, can becommunicated to a remote user as described more fully below.

According to embodiments of the present invention, all of theaforementioned items are included in the system illustrated in FIG. 2A.In alternative embodiments, less than all the aforementioned items areprovided. For example, in a specific embodiment, a single automationinput/output stack is provided on the automation table. Moreover, otherembodiments of the present invention are not limited to these items, butmay include additional items as will be evident to one of skill in theart. Merely by way of example, processing and reading apparatus, inaddition to or as a replacement for the FIDX and AIX are provided bysome particular embodiments. In these particular embodiments, analysisof fluorescence, thermal, chemical, visual, or other properties areutilized in an automated manner to perform experiments of interest. Ofcourse, one of ordinary skill in the art would recognize manyvariations, modifications, and alternatives.

The methods and systems provided according to the present invention willhave a variety of uses. Merely by way of example, embodiments of thepresent invention provide a communications system used to transmitinformation related to a protein crystallization process to a remoteuser. In a specific embodiment, the communications system includesemail, voice mail, instant messaging, paging, and/or SMS. Anotherembodiment according to the present invention provides systems andmethods that use a database to manage information related to processesperformed using microfluidic elastomeric chips, for example, informationrelated to a protein crystallization process.

FIG. 2B is a simplified schematic diagram illustrating another automatedprotein crystallization system according to another embodiment of thepresent invention. As illustrated in FIG. 2B, selected components asillustrated in FIG. 2A are provided, along with a sample and reagentdispensing robot. Automation table 2050, track robot 2052 witharticulated arm 2054 and an input/output stack for microfluidic chips,samples and reagents 2070 and thermal cycling device 2073 are providedaccording to an embodiment of the present invention. The platform isfabricated from a rigid material capable of maintaining the platform ina substantially stationary position, for example, with a spatialmovement of less than about 1 millimeter when the robot device operatesand moves the robot arm from a first spatial location to a secondspatial location. The input/output stack provides temporary storage forboth microfluidic chips, as well as a number of samples and reagents.The samples and reagents are utilized by the sample and reagentdispensing robot 2072 under computer control to dispense samples andreagents into appropriate chips. Therefore, the embodiment of thepresent invention illustrated in FIG. 2B provides the benefits availablethrough the system illustrated in FIG. 2A and additionally provides forautomated sample and reagent storage and dispense operations.

FIG. 2C is a simplified schematic diagram illustrating an automatedprotein crystallization experiment and analysis system according to anembodiment of the present invention. As illustrated in FIG. 2C, a liquidhandling system 2074, a Microfluidic Chip Automation System 1076, and aBackend Automation System 2078 are coupled to each other. In a specificembodiment, the Liquid Handling System 2074 comprises a reagent andsample storage, loading, and disposal system including a reagent andsample dispensing robot as illustrated in FIG. 2B. Moreover, in someembodiments, the Microfluidic Chip Automation System 2076 includes theelements shown in FIG. 2B. Thus, the system illustrated in FIG. 2Cincludes a number of sub-components in an integrated and automatedmanner. Furthermore, Backend Automation System 2078 generally includesmethods and apparatus to automate the process of removing crystals fromparticular chips and performing analysis, including x-raycrystallography. As will be evident to one of skill in the art, computerhardware and software (not shown) will be utilized in variousembodiments of the present invention to control the operation andinteraction of the several systems illustrated in FIG. 2C.

Transfer sections 2075 and 2077 are provided in FIG. 2C. Transfersection 2075 is adapted to move a microfluidic device between the LiquidHandling System 2074 and the Microfluidic Chip Automation System 2076.In some embodiments, transfer section 2075 is operated under computercontrol to provide for automated motion of microfluidic devices fromsystem to system. Transfer section 2077 is adapted to move amicrofluidic device between the Microfluidic Chip Automation System 2076and the Backend Automation System 2078. In some embodiments, transfersection 2077 is operated under computer control to provide for automatedmotion of microfluidic devices from system to system.

FIG. 2D is a simplified schematic diagram of system software accordingto an embodiment of the present invention. As illustrated in FIG. 2D, aLiquid Handling System 2080, a Microfluidic Chip Automation System 2082,and a Backend System 2084 are provided and coupled to Liquid HandlingServer Software 2086, a Microfluidic Chip Workflow Manager 2088, and aMicrofluidic Chip Backend Processor 2090, respectively. The LiquidHandling Server Software is coupled to a Sample and Reagent Database2092. The Microfluidic Chip Workflow Manager and the Microfluidic ChipBackend Processor are coupled to a Database 2094. In some embodiments ofthe present invention, the Sample and Reagent Database includes datautilized by the Liquid Handling Server Software to control and automatethe Liquid Handling System. In general, elements 2086 and 2092 may beprovided on one or more computers as appropriate to the particularapplication.

Experiment Manager and Crystal Vision software packages 2095, 2096, and2097 are coupled to the Database 2094 as illustrated in FIG. 2D. Asdescribed previously, users 2098, 2099, and 2099 a interact with thesoftware control system utilizing one or more instances of ExperimentManager and Crystal Vision software. Referring back to FIG. 2, thefunctionality provided by the system described with respect to FIG. 2 isincluded in the embodiments illustrated in FIG. 2D, as well asadditional functionality.

FIG. 2E is simplified schematic network diagram illustrating a computernetwork according to an embodiment of the present invention. Asillustrated, one or more computers are utilized in an embodiment tocontrol one or more hardware components. For example, referring to FIG.2A, computers A and B are coupled to the AIX and the FIDX, respectively.Both computers A and B are coupled to a computer network 2081. ComputersC and E are coupled to a robot, for example, the track robot with anarticulated arm, and the Microfluidic Chip Hotels, respectively.Computers F and G are coupled to the Database and the Workflow Manager,respectively. As illustrated, computers C, E, F, and G are also coupledto the computer network 2081. Accordingly, according to embodiments ofthe present invention, the computer network 2081 provides forcommunication of data and control commands between various hardware andsoftware components as described herein.

FIG. 2E also illustrates computer H coupled to an Auto Processor andcomputer G coupled to a Backend Processor. Accordingly, complete proteincrystallization experimental systems, represented by the Auto Processor,are coupled to particular dedicated pieces of equipment and software insome embodiments of the present invention. Merely by way of example, themethods and systems provided by the embodiments of the present inventionillustrated in FIG. 2E provide for combination of a number of FIDXsystems as appropriate to a particular application. One of ordinaryskill in the art would recognize many variations, modifications, andalternatives.

A number of user computers running the Experiment Manager and Crystalvision are also are coupled to the computer network as illustrated inFIG. 2E. As discussed in relation to FIG. 2, automated delivery ofexperimental conditions and information, as well as feedback processesadapted for the control, changing, and optimization of variousexperiments are provided by embodiments of the present invention.

Embodiments of the present invention provide a graphical user interface(GUI) through which users interact with the system. For example, anExperiment Manager, e.g., a TOPAZ™ Experiment Manager, is included insome embodiments as discussed in relation to FIGS. 1 and 2. Thefollowing section provides details related to the functionality of theExperiment Manager software and examples of methods of use andoperation.

FIG. 3 is a simplified diagram illustrating a graphical user interface(GUI) according to an embodiment of the present invention. Dependingupon the embodiment, the present GUI can be implemented using certainmethods and systems described herein. As an example, the system andmethod can be those generally described above, although others may alsobe used, depending upon the specific embodiment. In general, the GUIlayout includes a Menu Bar 310, a Tool Bar 320, a Library Explorer Panel330 and an Experiment Explorer Panel 332, a Status Bar 340 and aWorksheet panel 350, as shown in FIG. 3, which illustrates a particularembodiment of the present invention. The following functions areprovided by GUIs included within the scope of embodiments of the presentinvention.

Menu Bar 310—Top level menus include File, Edit, Settings, View, Tools,and Help. In some embodiments, each of the menus are pull-down menusactivated by an input device, for example, a mouse or a touch screen,and include a number of menu items generally accessible throughactivation of the pull-down menus. In particular embodiments, theselection of a menu item will result in an action, whereas in otherembodiments, the selection of a menu item, for example, a menu itemfollowed by ellipsis ( . . . ), will result in generation of a pop-upwindow.

File  Database Log-off  New (allows a user to create a new item ofReagent set (also referred to as  a screen), Target, Sample, and a ChipRun (Template and Ready-to-Run))  Save (allows a user to save thecurrent worksheet)  Save As (allows a user to save the current worksheetas a different data  file)  The availability of this operation isdependent on the kind of worksheet.  Additional details regardingworksheets are found in the worksheet  specification below.  Close(allows a user to close the current worksheet)  Close All (allows a userto close all the opened worksheets)  Import Chip Runs (allows a user toimport Chip Runs from acquired  outside database application)  Export(allows a user to export the data in the current worksheet to a  CSVfile)  History (allows a user to list the history of opened worksheet)The history  may include when and who created the data and when and wholast  modified the data.  Exit Edit Menu  Rename (allows the user torename the currently selected item in the  Explorer Panel if the dataitem is not referenced)  Delete (allows a user to delete the currentlyselected item in the Explorer  Panel if the data item is not referenced)Setting Menu  Classification Settings (Prompt the same dialog as the“Default Settings”  in the Crystal Vision to define the User and AutoClassification settings)  Generally, the Classification Settings aresystem wide, whereas the three  settings below are user specific.  ChipRun Result Worksheet Settings (Define the data reporting (e.g., what user and auto classification will be reported in a Chip Run result worksheet))  Target Result Worksheet Settings (Define the datareporting (e.g., what  user and auto classification will be reported ina Target result worksheet))  Query Result Settings (Define what type ofdata will be reported) View Menu  Show/Hide Tool Bar  Show/Hide StatusBar  Refresh Current Worksheet  Refresh Explorer  Refresh All Tools Menu Crystal Vision  Windows Explorer Window - List of the opened worksheetsand the current worksheet Help Menu  About Experiment Manager... Fluidigm on the Web  User Guide...

In alternative embodiments of the present invention, additional menusare provided, additional menus are provided in different orders, or oneor more menus are removed without departing from the scope of the claimsherein. Moreover, in certain embodiments, additional menu items areprovided, additional menu items are provided in different orders, or oneor more menu items are removed without departing from the scope of theclaims herein.

Embodiments of the present invention further provide a toolbar and astatus bar. The toolbar 320, as illustrated in FIG. 3, generallycontains the most used operations defined under the menus. The statusbar 340 generally contains information related to status and operationof the one or more programs. In an embodiment, the toolbar contains:

Toolbar   File New   File Save   Export Status Bar   Current User  Progress on the current operation   Date and Time

In alternative embodiments of the present invention, additionaloperations are provided on the toolbar, additional operations areprovided in different orders, or one or more operations are removedwithout departing from the scope of the claims herein. Moreover, morethan one toolbar is provided in certain embodiments, providing groupingsof operations as appropriate to the particular application.

Moreover, in some embodiments, worksheet GUI guidelines are provided. Ingeneral, there are two types of worksheets: worksheets for datamaintained by the system and worksheets for data maintained by the user.For an item, such as a component, displayed in the tabular form, thereis preferably a column indicating whether the item is being referencedor not. For a dataset, such as a Reagent Set, there is preferably afield indicating whether the data is being referenced or not. In aspecific embodiment, if the data is referenced, the attributes that canbe modified by a user are limited (depending what is the data item). Asdescribed more fully below, the detailed specifications for theworksheets are defined item by item.

In an embodiment, the Worksheet for Data Maintained by the Systemincludes: Reagent Components, Ligands, and Sample Components.Preferably, each of the worksheets contain the following: a table thatcontains the list of data items and a toolbar that contains tools forCreating, Removing, and Editing of a selected data item. The Worksheetfor Data Maintained by the User essentially includes all items exceptthe three described above.

Generally, a worksheet has the following generic form as illustrated inTable 1.

TABLE 1 Item Name: “ABC” (the name displayed in the Explorer panel) HighLevel Item Attributes, such as ID, part number, vendor, and the likeDetailed descriptions for the sub items, such as component formulationfor a reagent set, constructs for a target, and the like

As illustrated in FIG. 3, in embodiments of the present invention, twoexplorers are provided. As illustrated the Library Explorer providesinformation on the library: (e.g., reagents, targets and samples) andthe Experiment Explorer provides information related to the experimentalresults. Through selection of items in the Explorers, typically using amouse or like input device, menus and sub-menus are opened, closed, andmanipulated to display information, worksheets, input forms, outputdisplays, and the like. The examples provided below are not the onlyexplorer lists provided by the present invention, but are merelyexemplary. In the Explorers illustrated in the figures, various examplesof reagent sets or screens, targets, samples, and the like are shown forpurposes of demonstration. Of course, the specific items presented inthe Explorers according to embodiments of the present invention willdepend on the particular applications.

Library Explorer 330—In an embodiment of the present invention, LibraryExplorer provides information related to the Reagent or Screens, Targetand Sample. Merely by way of example the Library Explorer displays thefollowing list in a specific embodiment of the present invention:

± Reagent Sets (icon)   | -- Mix 1   | -- Mix 2   | -- Mix 3   | -- MixPEG   | -- .....   ± Design Templates (icon)     | -- Optimization #1    | -- Translation #1     | -- ..... ± Targets (icon)   | --alpha-lactalbumin   | -- beta-lactoglobulin   | -- catalase   | --glucose isomerase   | -- ..... ± Samples (icon)   | -- alpha-lactalbumin#1   | -- alpha-lactalbumin #2   | -- catalase #1   | -- .....   | --Ligands (icon)   | -- Cofactors (icon)   ± Sample Buffer SolutionTemplates (icon)     | -- Buffer #1     | -- Buffer #2     | -- ..... ±Reagent and Sample Buffer Components (icon) ± Dispensing Mapping (icon)  | -- 96 reagent plate to TOPAZ ™ Screening Chip   | -- 96 reagentplate to TOPAZ ™ Xray Chip   | -- 96 reagent plate to 96 well   | --.....

In operating the system, when a user selects (i.e., double clicks) anitem, the Experiment Manager software will display the informationcontent of the item as a worksheet in the worksheet panel. The examplesprovided in the Library Explorer illustrate above are not intended tolimit the present invention, but merely to provide examples ofinformation accessible through the Library Explorer according to anembodiment of the present invention. Additional items can be added,removed, or the order in which the items are displayed can be changedwithout departing from the scope of the claims herein.

Utilizing the Library Explorer, a user may add a new Reagent Set,Target, Sample, and Dispensing Mapping if desired. If an item is notbeing referenced by another dataset, the user will be able to remove ormodify an item from Reagent, Set, Target, Sample, and DispensingMapping. Once it is referenced by another dataset, a user can't removethe item. However, a user could perform limited modification to areferenced item. The limitation will be dependent on which item the usertries to modify, as will be explained below.

Experiment Explorer 332—In an embodiment of the present invention, theExperiment Explorer (sometimes also referred to as a Data Explorer)provides information related to the experiments performed using theautomated system. Merely by way of example the Experiment Explorerdisplays the following list in a specific embodiment of the presentinvention:

± Chip Runs (icon)   | -- st001 (active icon)   | -- st003 (active icon)  | -- st004 (complete icon)   | -- st005 (complete icon)   | -- st006(complete icon)   | ...   | -- st002 (Cancelled icon)   | -- ± Templates(icon)     | -- TOPAZ ™ #1     | -- TOPAZ ™ #2   | -- ± Ready-to-Run(icon)     | -- st0010     | -- st0011 ± Crystallization Results byTargets    | -- alpha-lactalbumin    | -- beta-lactoglobulin    | --catalase    | -- glucose isomerase    | -- ..... ± Query Reports    | --Mix 1 Query #1    | -- Target Query #1    | -- Target Query #2    | --.....

When a user selects (double clicks) an item, the Experiment Manager willdisplay the information content of the item as a worksheet in theworksheet panel. As illustrated in FIG. 3, the Components item isselected and the Components information, for example, ComponentA andComponentB, is displayed in the worksheet panel. The detailedspecifications of the GUI contents when a user selects an item in thelist are explained in the subsequent sub-sections below. The examplesprovided in the Experiment Explorer illustrate above are not intended tolimit the present invention, but merely to provide examples ofinformation accessible through the Experiment Explorer according to anembodiment of the present invention. Additional items can be added,removed, or the order in which the items are displayed can be changedwithout departing from the scope of the claims herein.

According to some embodiments of the present invention, a user maycreate, modify and delete a Template or Ready-to-Run chip run at anytime. Additionally, users are also be able to delete a chip run asdesired. In some embodiments, this deletion process involves the use ofa Cancelled icon.

Reagent, Target, and Sample User Interface Specifications are providedby embodiments of the present invention as described below. With respectto the reagent set worksheet, when a user double clicks on a reagent setitem in the Library Explorer, the Experiment Manager will display aReagent Set Worksheet for the selected reagent set. In the reagent setworksheet, there will be two pages (tabs). The default one will displayall the reagent set design information, and the second one will displaya list of the physical reagent sets.

FIG. 4 is a simplified diagram illustrating a reagent or screen userinterface (UI) according to an embodiment of the present invention. Asillustrated in FIG. 4, the layout for a reagent set design pageaccording to an embodiment of the present invention is shown. In thereagent or screen UI shown, a table listing one or more of the reagentplates plus their attributes is displayed. Additionally, a Library andExperiment Explorer are provided as discussed above. In a specificembodiment, the reagent set design page includes a listing of all thereagent plates. Information regarding the reagent attributes isdisplayed in the UI, including the name, creation information,modification information, part number, vendor, and a description.Alternative embodiments, provide additional information, remove one ormore information items, or present the information items in a differentorder without departing from the scope of the claims herein.

When a user selects a reagent plate (a row), the physical plateinformation will be displayed in a separate area and available formodification. A user will be able to add, remove, and modify a physicalplate. Once a physical plate is reference by some other data, it can nolonger be removed. There will be a generic reagent plate (default) for agiven reagent set. The user cannot remove this item from the list. In aspecific embodiment, if a reagent set does not include an informationitem, for example, Conductivity, Osmolality, or pH, that column will notbe displayed. In other words, the column will be displayed only if thereis data.

Depending upon the embodiment, the present methods and systems providecertain output formats, which are described in more detail below.Systems according to the present invention will also include Reagent andSample Buffer Components Worksheets. When a user double clicks on theReagent and Sample Buffer Components in the Library Explorer, theExperiment Manager will display the Reagent and Sample Buffer ComponentsWorksheet. In some embodiments of the present invention, the ComponentsWorksheet will provide a table displaying all the components used forall the reagent sets with the following attributes:

Name Reference - If an item is referenced or is a standard name foralias(s), a reference icon will be shown. Alias - If an item isreferenced as a standard name for other alias, the standard name iconwill be shown. If an item is an alias, an alias icon will be shown. Ifan item is not referred as the standard for an alias or it is not analias, nothing is shown in the alias column. Used for - The indicationof whether the component is used for reagent and/or sample. Reference IDDescription

As will be evident to one of skill in the art, the attributes displayedin the worksheets are not limited to this list. In alternativeembodiments, additional attributes are added, one or more attributes areremoved, or one or more attributes are provided in a different orderwithout departing from the scope of the claims herein.

A user will also be able to add a new component, remove a component (ifit is not referenced), or modify a component by using the Reagent andSample Buffer Components Worksheet. Additionally, in general, a userwill be able to modify the Description, and Aliases. A user could alsomodify the name for the component if it is not being referenced.Additionally, a user will be able to sort on any column.

In a particular embodiment, an Aliases sub panel will display thealiases information for various components (Components Alias) with theformat as illustrated in Table 2. A user will be able to add and removean alias using this sub panel.

TABLE 2 Standard Name Alias ABC ABC-1 ABC-2 ABC-3 ABC-4 ABC-5 ABC-6

Embodiments of the present invention provide a Component Stock Solutionsub panel with a table displaying the list of Stock Solutions with thefollowing attributes:

Part Number Vendor Component Name Concentration Concentration Unit pH

As will be evident to one of skill in the art, a Stock Solution is acomponent solution that a user can purchase from a vendor. Each StockSolution is associated with one component, and each component can havemultiple Stock solutions (with different concentration and pH). Asdescribed in more detail throughout the specification, a user can use asoftware wizard to create a stock solution or import multiple stocksolutions from, for example, a CSV file, as defined in the file formatsection.

Systems according to embodiments of the present invention will generallyalso include a Reagent Dispensing Mapping Worksheet. When a user doubleclicks on a Dispensing Mapping item in the Library Explorer, theExperiment Manager will display a Dispensing Mapping Worksheet. In aspecific embodiment of the present invention, this worksheet displaysthe following items:

Attributes Generally, there will be two sections or plates displayed, aReagent Plate section and a Chip/Plate - Topaz section. In the reagentplate (source) and chip/plate (destination), the graphics will indicatewhether a well is mapped (both in source and destination plate) or not.In a specific embodiment, all the wells in the destination chip/platewill be mapped. Additionally, there will typically be a list ofwell-to-well maps. When a user clicks on the list, the user will be ableto see the wells highlighted in both the source and destination plates.

As will be evident to one of skill in the art, the items displayed inthe worksheets are not limited to this list. In alternative embodiments,additional items are added, one or more items are removed, or one ormore items are provided in a different order without departing from thescope of the claims herein.

The Dispensing Mapping Worksheet will operate as follows in a particularembodiment according to the present invention. A user will not be ableto modify the name, reagent plate format, and chip run plate format oncethe dispensing mapping is referenced. The user can modify (e.g., correcta mistake) the mapping (well-to-well) after receiving a warning from thesystem. Generally, there are two modes in this worksheet: editing modeand view only mode. During operation in the view only mode, the user canonly view the mapping information.

Systems according to embodiments of the present invention will generallyalso include a Target worksheet or UI that typically includes threedisplay segments: Target Attributes; Constructs; and Samples. TheConstructs segment will provide a list of constructs with theirattributes. The user will be able to create, delete, and modify aconstruct through this UI. However, once a construct is referenced, itcannot be removed and user can only modify its sequence and descriptionattributes. Additionally, there will be a default construct (fulllength) for each target. The construct attributes according to anembodiment of the present invention are:

Name: Full Length Reference ID: Sequence: Same as the target.Description: Default construct for the target [name of the target].

A user will be able to modify the Reference ID and Description in someembodiments of the present invention. The full length construct cannotbe deleted from the list in some embodiments of the present invention.

The samples segment of the Target worksheet will generally contain alist of all the samples that contain the target. In an embodiment, thedisplay attributes are:

Name Barcode ID Construct

When a user selects (single clicks) on the sample tab in the proteintarget worksheet, a list of protein samples will be displayed thatcontain the protein target. Additional details are discussed in relationto the Sample worksheet below.

In an embodiment of the present invention, when a user double clicks onthe Ligand in the Library Explorer, the Experiment Manager will displaythe Ligand Worksheet or UI. In the Ligand Worksheet, there will be atable displaying all the ligands currently in the system with thefollowing attributes:

Name Reference ID Description

The user will also be able to add a new ligand, remove a ligand (if itis not referenced), or modify a ligand through this UI. The user willalso be able to modify the Reference ID, and Description. The user couldalso modify the name for the ligand if it is not being reference.

When a user double clicks on the Cofactor in the Library Explorer, theExperiment Manager will display the Cofactor Worksheet or UI. In theCofactor Worksheet, there will be a table displaying all the cofactorscurrently in the system with the following attributes:

Name Reference ID Description

The user will also be able to add a new cofactor, remove a cofactor (ifit is not referenced), or modify a cofactor through this UI. The userwill also be able to modify the Reference ID, and Description. The usercould also modify the name for the cofactor if it is not beingreference.

According to some embodiments, there will be two display sections forthe Sample Buffer Solution Template Worksheet or UI. The first part ofthe UI displays: Buffer Solution Name and Description. The second partdisplays a list of the following attributes:

Component Name Component Type Component Concentration ComponentConcentration Unit Component pH.

The user will also be able to create a new buffer solution, remove abuffer solution (if it is not referenced), or modify a buffer solutionby using this UI. The user will be able to modify the ID, andDescription. A user will also be able to modify the name for the buffersolution if it is not being reference.

In some embodiments, the Sample Worksheet or UI will have three displaysegments: Sample Attributes; Constructs, Cofactor, and Ligand; andSample Buffer Component. These segments include the following elements:

Sample Attributes Name Barcode ID Vendor (who made it) Part NumberDescription Number of constructs Number of cofactors Number of ligandsNumber of Components

Constructs, Cofactor, and Ligand—A list displays the construct, cofactorand ligands with the following attributes:

Constructs   Target Name/Construct Name   Construct Concentration  Construct Concentration Unit Cofactor   Cofactor Name   CofactorConcentration   Cofactor Concentration Unit Ligand   Ligand Name  Ligand Concentration   Ligand Concentration Unit

The graphic display for the Sample Worksheet will be similar to Table 3.Of course, one of ordinary skill in the art would recognize manyvariations, modifications, and alternatives.

TABLE 3 Type Name Concentration Unit Target/Construct Catalase/FullLength 25 mg/ml Cofactor ABC 30 mg/ml Ligand CDF 50 mg/ml

Sample Buffer Component—The Sample Buffer Component will include theSample Buffer Solution Template Name (if it exists) and a list ofcomponents with the following attributes:

Component Name Component Type Component Concentration ComponentConcentration Unit Component pH.

When a user creates a new sample, it can be created from, for example, aCSV file or it can be created by a new wizard. In an embodiment of thepresent invention, the wizard to create a new sample is constructed asfollows:

Identifiers   Name   Barcode ID   Vendor (who made it)   Part Number  Description Targets   Number of constructs   Target Name   ConstructName   Construct Concentration   Construct Concentration Unit Ligands  Number of Ligands   Ligand Name   Ligand Concentration   LigandConcentration Unit Cofactors   Cofactor Name   Cofactor Concentration  Cofactor Concentration Unit Buffer Solution   Select an existingbuffer solution Components   Number of Components   Component Name  Component Type   Component Concentration   Component ConcentrationUnit   Component pH

If a user selects an existing sample buffer solution template, theinformation will be filled in automatically. In the Worksheet, the userwill be able to manually enter all the required information. When a userselects the Target name, Construct name, Ligand Name, Cofactor Name andComponent Name, there will be a dropdown list box for available for thespecific data. When a user edits the Unit cell, there will be a dropdownlist, but user will be able to add a new unit.

Embodiments of the present invention provide a Chip Run interface. AChip Run Creation template is also provided, including the followinginformation:

Chip run name (also used as Chip run template name) CrystallizationMethod (e.g., TOPAZ ™, Vapor Diffusion) (Optional) Chip Type (e.g. 1.96,4.96, 8.96, X-ray chip or 96 microtiter plate) (Optional) Reagent Set(Optional) Reagent Dispensing Mapping (Optional) Incubation Temperature(Optional) Hydration Level (Optional) Destination file location(Optional) Number of scans and time (Optional)

A Chip Run Template may be created by using a wizard with the followingsteps:

Template Name Crystallization Method (e.g., TOPAZ ™, Vapor Diffusion)(Optional) Chip Type (e.g., TOPAZ ™ 1.96, 4.96, 8.96, X-ray chip or 96microtiter plate) (Optional) Reagent Set (Optional) Reagent DispensingMapping (Optional) Incubation Temperature (Optional) Hydration Level(Optional) Destination file location (Optional) Number of scans orimages and time (Optional)

Additionally, a Chip Ready-to-Run Template is created with the sameinformation as found in the as the Chip Run Template, except that allthe fields, except the number of scans and time, have to be defined. TheChip Ready-to-Run Template is created using a wizard with the followingsteps:

Initial Entries   Chip Run Name   Select a Chip Run Template (Optional)  Owner of the Chip Run Additional Entries   Same as the steps in theChip Run Template wizard, except that the   user has to enter all theinformation with the exception of the   Hydration level, which is stillOptional.

If a Chip Run template is selected, a user will still guided through allthe steps with the information pre-populated in some or all of thefields.

When a user double clicks on a Template item in the Experiment Explorer,a Chip Run template worksheet will be opened. All the Chip Run templateinformation will be displayed and editable. The display format willfollow the worksheet GUI guideline defined previously.

When a user double clicks on a Ready-to-Run item in the ExperimentExplorer, a Chip Run Ready-to-Run worksheet will be opened. All the ChipRun Ready-to-Run information will be displayed and editable. The displayformat will follow the worksheet GUI guideline defined previously.Essentially, the Ready-To-Run and Template worksheets are the same,except the title of “Name” part. For the Template, it is “Chip RunTemplate Name.”. For the Ready-To-Run, it is the “Chip Run Name.” Inthis worksheet, a user will be able to save a Ready-to-Run to a HTMLfile or View the HTML file with the Internet Explorer (IE) and thenprint the HTML file with IE. The purpose for this HTML file is for userto follow the instructions to prepare and to run the chip.

When a user double clicks on an Active, Completed or Cancelled icon inthe Experiment Explorer, a Chip Run worksheet will be opened. In aspecific embodiment, this worksheet contains two display sections. Thefirst section displays the complete chip run information defined in the“Chip Run Information.” The second section displays the summaryexperiment results, presented in the format illustrated in Table 5.

TABLE 5 Chip Crystallization Sam- Reagent Day Day Day Day Day StartedDura- Result Run Method ple Reagent Set 1 2 3 5 7 Overall On tion

 Crystal A ST001 Topaz SA1 OptiMix 1: #1 OptiMix 1 1 1 Nov. 30, 2004 6days Screening

 Crystal A ST001 Sitting Drop SA2 OptiMix 1: #2 OptiMix 1 1 1 Dec. 1,2004 7 days

 Crystal B ST001 Topaz SA1 OptiMix 1: #3 OptiMix 1 1 1 Dec. 2, 2004 5days Screening

 Crystal B ST001 Topaz SA1 OptiMix 1: #4 OptiMix 1 1 1 Dec. 3, 2004 8days Screening

 Crystal B ST001 Sitting Drop SA3 OptiMix 1: #5 OptiMix 1 1 1 Dec. 4,2004 3 days

 Crystal C ST001 Sitting Drop SA4 OptiMix 1: #6 OptiMix 1 1 1 Dec. 5,2004 2 days

The report settings are defined by the “Chip Run Worksheet” Settings.The Crystal A, Crystal B, and Crystal C are examples of userclassification. The actual display will depend on the userclassification defined in the database. The color of the circles (foruser classification) or squares (for auto classification) will bedetermined from the classification settings defined in theClassification Setting.

A variety of operations are available using this worksheet. For example,a user will be able to sort on any column. When a user sorts the Resultcolumn, the sorting order will following the User Ranking defined by theClassification Setting. The user will also be able to print this worksheet (including both display sections). Other operations include:

Select a row and launch Crystal Vision for viewing the image for thegiven condition. Select a Sample and open the Sample Worksheet. Select aReagent and open the Reagent Set Worksheet. Select any number of rowsand generated a query results (See Experiment Result Query for details).

According to embodiments of the present invention, the user will be ableto select multiple chip runs in the Experiment Explorer, and then createa Multiple Chip Run worksheet. This worksheet displays a chip runsummary result table defined as illustrated in Table 6.

TABLE 6 # of Chip Crystallization Chip Reagent Auto # of # of # ofStarted Duration Run Method Type Sample Set Crystal Crystal A Crystal BCrystal C On (Days) ST001 TOPAZ ™ TOPAZ ™ SA1 OptiMix1 3 2 0 0 Nov. 1,2004 6 Screening 4.96 ST001 Sitting Drop TOPAZ ™ SA2 OptiMix1 3 2 0 0Nov. 1, 2004 6 4.96 ST001 TOPAZ ™ TOPAZ ™ SA3 OptiMix1 3 2 0 0 Nov. 1,2004 6 Screening 4.96 ST001 TOPAZ ™ TOPAZ ™ SA4 OptiMix1 3 2 0 0 Nov. 1,2004 6 Screening 4.96 ST002 Sitting Drop TOPAZ ™ SA5 OptiMix2 4 1 3 Nov.1, 2004 7 1.96 ST003 Sitting Drop Wall Plate SA6 OptiMix4 3 3 Nov. 2,2004

The report settings are defined by the “Chip Run Worksheet” Settings.The Crystal A, B, and C are examples of user classifications. The actualdisplay will depend on the user classifications defined in the database.There can be additional User and System information reported, as definedby the Chip Run Worksheet Settings. The operations for this worksheetare the same as the ones defined in the Single Chip Run worksheet.

When a user double clicks on a Target result in the Experiment Explorer,a Target Result worksheet will be opened. This worksheet will containtwo display sections. The first section displays the target information(without the constructs). The second section displays thecrystallization results in the format illustrated in Table 7.

TABLE 7 Crystal- Crys- Crys- Crys- Sam- Reagent lization tal A tal B talC Chip Started Dura- Target Construct Ligand Cofacert ple Reagent SetMethod At At At Run On tion BSGCAIR30322 Full length SA 1 Reagent #1OptiMix 1 Topaz 2.5 ST001 Nov. 11, 6 days Screening 2004 BSGCAIR30322Full length SA 2 Reagent #1 OptiMix 1 Sitting 3.5 ST004 Nov. 12, 7 daysDrop 2004 . . . . . . . . . . . .

The report settings are defined by the “Target Result Worksheet”Settings. The Crystal A, B and C are examples of user classification.The actual display will depend on the user classification defined in thedatabase. Operations for this worksheet include:

Sort on any column. Print this worksheet (including both displaysections). Select a row and launch Crystal Vision for viewing the imagefor the given condition. Select a Sample and open the Sample Worksheet.Select a Construct and open the Target Worksheet. Select a Ligand andopen the Ligand worksheet. Select a Cofactor and open the Cofactorworksheet. Select a Reagent or a Reagent set and open the Reagent SetWorksheet. Select a Chip run and open the Chip Run Worksheet. User willbe able to select any number of rows and generated a query results (SeeExperiment Result Query for details).

The user will be able to select multiple targets in the ExperimentExplorer, and then create a Multiple Target worksheet. The multipletarget worksheet displays the crystallization result table, which isidentical to the table defined in the Single Target Worksheet. Therewill be multiple targets in the table.

Embodiments of the present invention provide for formatting of databasequery results. For example, when a user perform an experiment querythrough any mechanism, the following information will be displayed:

Sample Information   Sample Name   Sample Barcode ID (physical if any)  Target(s)   Construct(s)   Construct Concentration(s)   ConstructUnit(s)   Ligand(s)   Ligand Concentration(s)   Ligand Unit(s)  Cofactor (s)   Cofactor Concentration(s)   Cofactor Unit(s)   SampleComponent     Component Name(s)     Component Concentration(s)    Component Unit(s)     Component pH(s) Reagent Information   ReagentName   Reagent Set Name   Reagent Plate Barcode ID (physical if any)  Reagent pH   Reagent Conductivity   Reagent Osmolality   Formulation    Component Name(s)     Component Concentration(s)     ComponentUnit(s)     Component pH(s) Chip Run Information   Chip run name (alsoused as Chip run template name)   Chip Type (e.g., TOPAZ ™ 1.96, 4.96,8.96, X-ray chip or   96 microtiter plate)   Crystallization Method(e.g., TOPAZ ™, Sitting Drop, Hanging   Drop, Microbatch and Other)  Incubation Temperature   Hydration Level   FID Time   DurationExperiment Result   User Classification #1 At (days)   UserClassification #2 At (days)   User Classification #3 At (days)   . . .(the number of User Classification will depend on the number of   userclassification available)   Auto Crystal At (days)

In the “Query Result Settings” dialog, the user can customize the formatwith which data is reported. For example, a user can remove all the ChipRun information and the Sample Component information. Of course,depending on the particular application, a user will preferentiallyselect the information to be displayed.

The Query Report Worksheet contains two display sections. The first partdisplays the report name, and Query criteria used for the query. Thesecond part displays the experiment results for each conditions found.The table header will consist of three rows for the required displayhierarchy. One operation provided is the ability to export the data to aCSV file.

For a predefined query, at the end of the query, as defined below, aquery report worksheet will be generated. For example, a Reagent SetData Analysis Wizard according to an embodiment of the present inventionis provided as follows:

Select a reagent set. Define the Chip Run criteria (AND relationship)  Dates   Targets   Crystallization Method   Experiment Parameter (FID,Hydration level, and incubation   temperature) Define the interestedexperiment results (User calls and Auto calls) Find all the reagentsmeeting the criteria defined in Step #1. Find all the chip runscontaining the reagents found in Step #4 and also meet the criteria in2. Define an Analysis Report Name. Default as “Untitled #1, . . . Createa Query Report Worksheet.

In some embodiments, the last four steps are automatic, with no userintervention required.

Additionally, a Reagent Data Analysis Wizard according to an embodimentof the present invention is provided as follows:

Select a reagent or a number of reagents. Choose whether other reagentswith same composition but different concentration and pH will be searchor not. Define the Chip Run criteria (AND relationship)   Dates  Targets   Crystallization Method   Experiment Parameter (FID,Hydration level, and incubation   temperature) Define the interestedexperiment results (User calls and Auto calls) For all the reagents withsame composition (precipitate and salt) but with different concentrationand pH if the option is chosen. Find all the chip runs containing thereagents found in 4 and also meet the criteria in 2. Define an AnalysisReport Name. Default as “Untitled #1, . . . Create a Query ReportWorksheet.

In some embodiments, the last four steps are automatic, with no userintervention required.

Additionally, a Component Data Analysis Wizard according to anembodiment of the present invention is provided as follows:

Select a component or a number of components. Define the concentrationrange for each component. User can add pH range as a constraint as well.Define the Chip Run criteria (AND relationship)   Dates   Targets  Crystallization Method   Experiment Parameter (FID, Hydration level,and incubation   temperature) Define the interested experiment results(User calls and Auto calls) For all the reagents containing thecomponent(s). Find all the chip runs containing the reagents found in 4and also meet the criteria in 2. Define an Analysis Report Name. Defaultas “Untitled #1, . . . Create a Query Report Worksheet.

In some embodiments, the last four steps are automatic, with no userintervention required.

Additionally, a Component Sub-Name Data Analysis Wizard according to anembodiment of the present invention is provided as follows:

Define a component sub-name. Define the concentration range for thesearched component(s). User can add pH range as a constraint as well.Define the Chip Run criteria (AND relationship)   Dates   Targets  Crystallization Method   Experiment Parameter (FID, Hydration level,and incubation   temperature) Define the interested experiment results(User calls and Auto calls) For all the components containing thesub-name, and then find all the reagents containing the components. Findall the chip runs containing the reagents found in 4 and also meet thecriteria in 2. Define an Analysis Report Name. Default as “Untitled #1,. . . Create a Query Report Worksheet.

In some embodiments, the last four steps are automatic, with no userintervention required.

For a custom database query, the user will be able to:

Select a list attributes from reagent, target/construct, ligand,cofactor, sample, and chip runs, Define the criteria for each attributeDefine how to combine these attributes (AND or OR)

A Reagent Set Template Worksheet consists of three display sections,similar to a reagent set. The top left section describes the ReagentTemplate Attributes and includes:

Template Name Number of Partitions Attributes for Each Partition(concentration and pH changes for component or component type)Description

The top right section displays a 96 well plate graphics, such as thegraphic illustrated in FIG. 5. The bottom section displays thedispensing scheme for the components as illustrated in Table 8. In aspecific embodiment, Table 8 contains rows for 12 samples, but this isnot required by the present invention.

TABLE 8 Well Component #1 Component #2 Component #3 Water A1 10 20 30 10A2 A3 A4

Additionally, a Reagent Set Template Creation Wizard according to anembodiment of the present invention is provided as follows:

Define the Template Name Define the number of region, the size of eachregion and the location of each region. Define the number of componentsor number of component types. Iterate through each region, define thecomponent or component type is used for each region, and how theconcentration or pH changes required. The change can be linear or log.

Additionally, a Reagent Set Creation from a Reagent Set Design TemplateWizard according to an embodiment of the present invention is providedas follows:

Identify the reagent components. The components can be defined from acrystallization condition. Select a Reagent Set Design Template Mappingthe components to the components in the template. If the template isdefined using component type, this step is not needed. Find theComponent Stock Solutions required for the components. Calculate thedispensing from Component to reagent formulation. Create a new reagentset.

Embodiments of the present invention provide for a variety of systemsettings. For example, classification settings are provided using thesame dialog as the “Default Settings” in the Crystal Vision to definethe User and Auto Classification settings. Additionally, Chip Run Resultsettings are provided to define the data reporting, including what userand auto classification will be reported in a Chip Run result worksheet.In this setting dialog, the user will be able to select theclassification types, which will be used for both Single Chip Run andMultiple Chip Runs worksheets as follows:

  Classification Crystal (Auto Classification) User Classification #1(Say, Crystal A) User Classification #2 (Say, Crystal B) ...

For the Multiple Chip Run worksheet, additional Experiment Parameters,User information and System Information can be selected as follows. Insome embodiments, the User Information and System Information are onlyprovided for the Multiple Chip Runs Worksheet.

  Experiment Parameter  Incubation temperature  Hydration level  FIDTime User Information  Owner of the chip Run  Annotated by  Approved byStatus System Information  FID System Information  Imaging SystemInformation

The User Classifications are all the user classifications defined in theClassification Settings. The default setting is the Crystal of AutoClassification and all the User Classifications of Crystal Type.

Target Result Settings are provided to define the data reporting,including what user and auto classification will be reported in a Targetresult worksheet. In this setting dialog, user will be able to selectthe classification types and user information as follows

  Classification  Crystal (Auto Classification)  User Classification #1(Say, Crystal A)  User Classification #2 (Say, Crystal B)  ... UserInformation  Owner of the chip Run  Annotated by  Approved by  Status

The default setting is the Crystal of Auto Classification and all theUser Classifications of Crystal Type.

Query Result Settings are provided to define the type of data that willbe reported in some embodiments of the present invention. There will bea list (tree view) with a “Check Box” for each item defined in the“Database Query Result Format” section.

A user may use the Import Chip Run function to import data acquiredoutside the Database Application. For example, data acquired duringperiods prior to the use of the Database Application by a user may beimported using this function. An Import Chip Run Wizard according to anembodiment of the present invention is provided as follows:

Select the import type: CSV file or Chip Run.

An Import Chip Run Wizard from, for example, a CSV file is also providedas follows:

  Select a CSV file Import all chip runs defined in the CSV file.

An Import Chip Run Wizard from Chip Run is further provided as follows:

Select a Chip Run - The wizard will automatically determine the numberof samples required for the chip run from an experiment INE file.

In a specific embodiment of the present invention, if the sample doesnot exist in the database, the wizard will automatically create asample. However, in this embodiment, the reagent set and the reagentdispensing mapping must already exist in the database. The number ofsamples defined will match the number of sample inlets in the chip run.If a user does not enter a sample for a given inlet, a blank sample willbe created.

Preferably, the database will hold at least 4 million experiments (e.g.,10,000 4.96 chip runs) with system performance measured by the followingmetrics:

 Response time for retrieving a data item in the Library Explorer will preferably be less than 1 second.  Response time for retrieving thecrystallization result for a given chip run  will preferably be 10second or less for the response time. Response time for retrieving thecrystallization result for a given target will preferably be linearlyrelated to the number of chip runs performed with the given target.

Additionally, users will be allowed to open multiple instances of theExperiment Manager on different computers at the same time. In anembodiment of the present invention, the Experiment Manager will use thefirst come/first served principle for the write access on any data. Insome embodiments, an optimistic update (or protected) write model willbe utilized. In this model, when data is opened, a time stamp will becreated for the opening of the data, thereby noting the last time thedata has been modified. Subsequently, when a write command is received,a comparison will be made between the time stamp for the open operationand the time stamp associated with the data at the time of the writeoperation. If the time stamps are equal (meaning the data has not beenmodified since the open operation), then the update will beaccomplished. If the time stamps are not equal, then the data updatingwill not be performed, in order to not overwrite the modifications madesince the open operation was performed. The same principle will beapplied to other software components (e.g., software associated with theFID Crystallizer) as well. In general, only one Experiment Manager willhave the write access to the Database at any one time.

FIG. 5 is a simplified diagram illustrating a Screen Plate Creation GUIaccording to an embodiment of the present invention. Information relatedto the reagents or screens is entered and provided in this GUI,including information related to salts, precipitants, and buffers. In aspecific embodiment, the concentration variation for a particular salt,precipitant, and/or buffer is illustrated both graphically andnumerically. As illustrated in FIG. 5, a color coded template 510illustrates the variation of salt and precipitant concentration as afunction of position over a plate. Section 520 of the GUI is utilized bya user to view and/or modify the variables in a particular region of theplate, for example Regions 1 through 4. The components of the selectedsalts, precipitants, and buffers are illustrated with the start and endpercentage change, along with the actual variation measured in physicalunits.

Merely by way of example, section 520 illustrates the salt sodiumacetate varying in concentration from 40% to 100% over a region as thesalt concentration in a row is incremented from 0.16 M to 0.4 M.Likewise, the precipitant 1,4-butanediol is varied in concentration from40% to 100% over the same region as the precipitant concentration in acolumn is incremented from 6 to 15% volume/volume. Selection of variousregions in the GUI will provide for viewing and/or modification of thevariables selected for that particular region.

As discussed previously, embodiments of the present invention provide aScore Card utilizing a GUI. The following functional specifications anddescription of the Score Card, for example, a TOPAZ™ Score Card, isprovided as an exemplary embodiment. As illustrated in FIGS. 6A and 6B,the figures are not drawn to scale, but merely represent the style ofthe GUI layout, not any particular dimensions associated with a specificGUI layout for the Score Card.

FIGS. 6A through 6D are simplified diagrams illustrating additionalgraphical user interfaces according to an embodiment of the presentinvention. FIG. 6A illustrates an example of a GUI layout for the ScoreCard according to an embodiment of the present invention. Asillustrated, a menu 610, tool bar 620, and a graphic/annotation area 630are provided. In the graphic/annotation area 630, a user can entercomments for a selected well or cell in the comment box 630 a or in thetable 630 b directly. FIG. 6B illustrates an example of a GUI layout foran Overall Score Card for microfluidic chips, (e.g., TOPAZ™ Chips)according to an embodiment of the present invention. As illustrated, anumber of TOPAZ™ Chips may be displayed on this Overall Score Card.

In the embodiment illustrated in FIGS. 6A and 6B, the Score Cardsprovide a number of menus 610. In alternative embodiments of the presentinvention, additional menus are provided, additional menus are providedin different orders, or one or more menus are removed without departingfrom the scope of the claims herein. Moreover, in certain embodiments,additional menu items are provided, additional menu items are providedin different orders, or one or more menu items are removed withoutdeparting from the scope of the claims herein.

In a specific embodiment of the present invention, the menus 610 includethe following top level menus:

  File Edit View Tools Help

The File menu includes the following functions according to anembodiment of the present invention:

Database Log-off Open - Open a chip run from a microfluidic deviceDatabase Save - Save the current chip run into one or more memories of acomputing system Export... - Export the data in the current worksheetto, for example, a CSV file. Exit

The Edit menu includes the following functions according to anembodiment of the present invention:

  Create a New Session Allow user to create a new “scan” session. ClearClear All

The View menu includes the following functions according to anembodiment of the present invention:

Classification Settings... Prompt the same dialog as the “DefaultSettings” in the Crystal Vision to view the User ClassificationSettings. Show/Hide Tool Bar Show/Hide Status Bar Refresh

The Tools menu includes the following functions according to anembodiment of the present invention:

  Notepad Paint Brush Windows Explorer

The Help menu includes the following functions according to anembodiment of the present invention:

  About Score Card... Fluidigm on the Web User Guide...

The toolbar 620 contains the most used operations defined under themenus. In a specific embodiment, the toolbar will contain:

  File Open File Save Export

In some Score Cards, a status bar 650 contains the following informationaccording to an embodiment of the present invention:

  Current User Date and Time

Referring to FIGS. 6A and 6B, the layout 642 and annotation 660 sectionsof the Score Card GUI are utilized as follows. When a user views aspecific microfluidic chip, for example, a TOPAZ™ Chip, the well plategraphic 640 will be replaced by a microfluidic chip graphic layout 642as illustrated in FIG. 6B. In some embodiments, the other elements inthe Score Card GUI remain unchanged when the microfluidic graphic layout642 replaces the 96 well graphic display 640.

In utilizing the Score Card, no explicit scan session is provided as inthe Image Acquisition process. However, a user can still create a listof manual scan sessions in order to keep track of the experiment resultover time. For example, a user can create a manual scan session on a perday basis, and examine the chip run and enter the crystallization of thechip run on a per day basis. Embodiments of the present inventionprovide for communicate between the Score Card and the Database 250through the Database Application Server 252.

FIG. 6C is a simplified diagram illustrating an Experiment Manager GUIaccording to another embodiment of the present invention. In this GUI, amenu bar 610, a toolbar 620 and a status bar 650 are provided asdiscussed above. Additionally, a Library and Experiment Explorer areprovided. In the worksheet area 640, a reagent dispensing mapping isillustrating, including a mapping for a reagent plate as well as amicrofluidic chip, for example, a TOPAZ™ chip. Selection by a user of aninlet, for example, inlet 1, will result in the display of the reagentwell, for example, reagent well A1, mapped to that inlet. The dispensingmapping may be both viewed and edited through this GUI by clicking on awell location in the Reagent Plate and then clicking on a reagent inlet(or well) to define the mapping.

FIG. 6D is a simplified diagram illustrating an Chip Run GUI accordingto yet another embodiment of the present invention. The Results tab ofthe Chip Run Worksheet displays a variety of information related to theresults of the Chip Run. In a specific embodiment, a Chip Run ResultsSummary section displays information including the reagent or screenname, the crystallization method, the chip type, and the Dispensing Mapamong other information. Moreover, a section including pull-down menusprovides for the user to select the particular information that will bedisplayed using the interface. Protein crystallization process results,including crystal ranking along with information related to theconditions of the protein crystallization process (e.g., protein sample,reagent, components and concentration of the salt, and the like) aredisplayed for viewing by the user.

Embodiments of the present invention provide for enhancements to the FIDCrystallizer (FIDX) as part of the Database Application. For example, auser is able to connect/disconnect the FIDX from the Database. Oneexample of an operation provided by embodiments of the present inventionis that when the FIDX is connected to the database, the user list willbe provided by the database, not the local FID database.

Additionally, when the FIDX logs in to the Database and a user performsa Reagent and Sample loading process, the FIDX will perform a check tosee whether the chip is already associated with a Chip Run or not asoutlined in an embodiment according to the present invention by thefollowing steps:

  1. If yes, proceed to perform the loading and update the Database withthe FID system information. 2. If no:  Query the Database to get the“Ready-to-Run” list, and select  a chip run from the list. If the chiprun was not created in advance,  ask the user to create a chip run forthe chip. Go to 1.

After performing these steps, the chip will be associated with a ChipRun in the Database. After performing the FID, the FIDX will update theDatabase with the FID time and FID system information.

Due to the multi-tasking nature of the FIDX as provided in embodimentsof the present invention, there is no explicit user login and logofffrom Database. The FIDX will automatically perform a Database login justbefore a user starts the Loading and FID processes. Whether a user needsto enter his/her password will depend on the User Administrationsettings. The software will communicate with the Database throughDatabase Application Server.

Embodiments of the present invention provide for enhancements to theImage Acquisition system as part of the Database Application. Forexample, a user is able to connect/disconnect the Image Acquisitionsoftware from the Database. One example of an operation provided byembodiments of the present invention is that when the Image Acquisitionsoftware is connected to the database, the user list will be provided bythe database, not the local user ID list from the barcode identifierlog.

Before an image scan is performed, the chip will already be associatedwith a Chip Run in the Database. Accordingly, a user will only need toinput the chip barcode and the User ID. After the Scan is completed, theImage Acquisition software will automatically update the Chip Run withthe information about the scan. The information will include:

  Time of the Scan Imaging System Information Status of the Scan:Scanned.

Similar to the FIDX, for the Image Acquisition software, there is noexplicit user login and logoff from Database. The Image Acquisitionsoftware will automatically perform a database login just before userstarts the scan. Whether user needs to enter his/her password willdepend on the User Administration settings. This software willcommunicate with the Database through the Database Application Server.

In some embodiments of the present invention, the Auto Processor, forexample, a TOPAZ™ Auto Processor, is provided with enhancements as partof the Database Application. The user will be able to connect/disconnectthe Auto Processor software from the database. When connected to thedatabase, user has to login/off to/from the Database. This software willcommunicate with the Database through the Database Application Server.

In a specific embodiment, the Auto Processor will perform databaseinformation updating. After a section is processed, the Auto Processorwill generally perform the following:

Perform a Database check to see whether the scan is defined in theDatabase or not. If yes, update the Database with the followinginformation:  Scan:   Auto Processor Software Version   Status: Changeto be Processed  Diffusion Experiment:   Ranking   Auto Call

In addition to other enhancements, Crystal Vision is provided withenhancements as part of the Database Application. In an embodiment, theuser will be able to connect/disconnect the Crystal Vision software fromthe database. When connected to the database, user has to login/offto/from the Database. When the Crystal Vision connects to the Database,the File—Open command will explore the Chip Runs defined in theDatabase, instead of in the File system.

Moreover, the Crystal Vision software will perform a databaseinformation update function in an embodiment of the present invention.In performing this function, the following information regarding thechip run will be updated by the Crystal Vision software:

Annotated by: Approved by: Last Modified at: User Calls, Flags forReview, and Comments of all diffusion experiments.

In some embodiments, there will be a mechanism for the ExperimentManager to instruct Crystal Vision to open a specific chip run and toinitialize the display for a specific diffusion experiment. This can beaccomplished through a Windows message. This software will communicatewith the Database through the Database Application Server.

Embodiments of the present invention provide a Database ApplicationServer 252 as illustrated in FIG. 2. In a particular embodiment, theDatabase Application Server is a COM server installed in the clientcomputer.

In some embodiments of the present invention, as discussed in relationto FIG. 2, a Publisher (sometimes referred to as Database Publishersoftware) is provided. In a specific embodiment, the Publisher is aTOPAZ™ Database Publisher software package. In a particular embodimentof the present invention, the Database Publisher software is installedalong with the Database in the same computer. The Database Publisher isa program utilized to publish the experimental results in a suitableformat. In a particular embodiment, the experimental results arepublished as HTML forms, which are saved to a designated Web Serverlocation.

The Publisher performs a variety of operations. These operations includeHTML Report Generation. Merely by way of example, the HTML reports aregenerated in an embodiment of the present invention in three differentreport formats as follows:

Ready-To-Run Chip Runs Report - This report provides a list of chip runsthat are in the Ready-To-Run state. The report is displayed in a HTMLform. When a user clicks a chip run (e.g., presented as a hyperlink),the Publisher displays the details of the selected ready-to-run chip runin another HTML form, which is the same as the one defined in thesection above regarding the Chip Run Ready-To-Run Worksheet. One purposeof this HTML form is for the user to view (and print out) the new chipsto be run. Chip Run Report - This report is an HTML table reporting theresults of all the chip runs from specified dates, which is defined bythe publisher setting dialog. The result format in this table isidentical to the table of a multiple chip run worksheet. Target Report -This report is an HTML table reporting all the results of all thetargets, which is defined by the publisher setting dialog. The resultformat in this table is identical to the table of a multiple targetworksheet.

The Publisher will generally have a series of user and system definedsettings. For example, in a particular embodiment, there are threesettings for the Publisher as follows:

Scheduler for report generation. The scheduler provides informationrelated to the timing with which reports are generated (e.g. whenreports are generated). Chip Run Result Format and Period. The resultformat setting is identical to the result format setting for the chiprun worksheet. In addition, a user can specify to report only those chipruns from certain period (e.g., last three months). Target ResultFormat. The result format setting is identical to the result formatsetting for the target worksheet. In addition, user can specify toreport only those chip runs from certain period (e.g., last threemonths).

The Database User Administrator software is installed along with theDatabase in the same computer. This is a program to manage users inrelation to database access. This program will manage the following userinformation, as discussed previously in relation to the Database.

  User Full Name User Description User Login Name User Password AccessRight. In some versions, only full access rights are supported. Status(either Active or Inactive)

Generally, the program performs a variety of operations including thefollowing:

  Add a new user with required attributes. Remove a user if it is notreferenced. Change the attributes of a user.

Moreover, there will be a setting indicating whether it is necessary fora user to enter his/her password when logging in to the Database foreach of the following applications:

  Experiment Manager FID Crystallizer Image Acquisition Crystal VisionScore Card

Embodiments of the present invention provide a number of possible SystemConfigurations. In a specific embodiment, the Database Applicationsupports both a Microsoft SQL server and an MSDE Server. The MSDE serveris the default configuration for Database Application, and the user isprovided with means to upgrade to an SQL server.

Several operating systems are compatible with embodiments of the presentinvention. Operating systems for the database include:

  Windows 2003 Server for Microsoft SQL Server Windows XP + SP2 forMicrosoft MSDE Server Windows 2000 + SP4 for Microsoft MSDE Server

Additionally, operating systems for all the Software Applicationsinclude:

  Windows 2003 Server - Compatible with the following Applications: Experiment Manager  User Administrator  Auto Processor  Crystal VisionWindows 2000 + SP4 (Compatible with all Applications) Windows XP + SP2(Compatible with all Applications)

In some operating systems, a Start Menu is provided. In these operatingsystems, the program listing is provided in the Start Menu as:

  Start →  All Programs →   Fluidigm →    Experiment Manager   Publisher    User Administrator    Score Card    FID Crystallizer   Image Acquisition    Auto Processor    Crystal Vision

Embodiments of the present invention provide Recommended SystemConfigurations. For the Experiment Manager Application, the recommendeddesktop display has a pixel count greater than or equal to 1280×1024pixels. The recommended display font is a normal font (96 dpi). Theminimum requirement for certain embodiments of the present invention isa desktop display with a pixel count greater than or equal to 1024×768pixels with a normal display font (96 dpi). The recommended process isan Intel® Pentium® 4 (1.5 GHz) or higher. The recommended available harddisk storage space is 2 GB of free memory.

For the Score Card Application, the recommended system configuration isthe same as for the Experiment Manager. For the Database, therecommended system configuration is an Intel® Pentium® 4 processor (1.5GHz) or higher and 200 GB of available hard disk space.

Embodiments of the present invention provide an applicationconfiguration for the automated system, which has been described herein.In a specific embodiment, the configuration includes the followingelements:

Database Computer:   Database User Administrator   Publisher (scheduledrunning during the off-hours)   Microsoft SQL or MSDE server + Database.Chip Run Storage and Processing Computer:   Microfluidic Chip AutoProcessor (scheduled running daily during the   off-hours)   All theMicrofluidic Chip Runs (image data).   Crystal Vision (Optional) FIDXComputer:   FIDX Crystallizer   Experiment Manager (Optional). Theprimary use case for Experiment   Manager on this computer is to designchip runs. It is not designed as   a general purpose data analysis andquery station due to the ergonomic   setup of the laptop. AIX Computer:  Image Acquisition   Experiment Manager (Optional)   Crystal Vision(Optional) Application Computer(s):   Experiment Manager (Optional)  Crystal Vision (Optional)   Score Card (Optional)

In some embodiments, the applications and chip run data are both placedon the Database computer. In other embodiments, which are provided forapplications characterized by heavy network traffic, the applicationsand chip run data are separated and stored on the Database Computer aswell as one or more separate computers.

In general, an installation procedure will be followed when installingthe software. When the installation procedure is started, the user ispresented with a choice of which applications the user intends toinstall or uninstall. Typically, there are two installation CD, forexample, TOPAZ™ Database Installation CDs. In a specific embodiment, thefirst CD is a TOPAZ™ Database CD, including the following softwarecomponents:

  Microsoft MSDE Server TOPAZ ™ Database TOPAZ ™ User AdministratorTOPAZ ™ Publisher TOPAZ ™ Database Application Server

Generally, all of the above software components will be installedautomatically. Additionally, in this specific embodiment, the second CDis a TOPAZ™ Database Application CD, including the following softwarecomponents:

TOPAZ ™ Experiment Manager TOPAZ ™ Score Card

Embodiments of the present invention provide an Automated System forProtein Crystallization. The system comprises a number of components,including:

Microfluidic Chips Microfluidic Chip FID Crystallizer (FIDX)Microfluidic Chip AutoInspeX Workstation (AIX) Transfer Robot Barcodereader Microfluidic Chip Hotel Microfluidic Chip User Input/OutputMicrofluidic Chip Automation Input/Output Microfluidic Chip AutoProcessor Crystal Vision Software Microfluidic Chip Experiment ManagerMicrofluidic Chip Workflow Manager Microfluidic Chip Database ServerComputers connected by network

Some embodiments of the present invention include all of theaforementioned items, whereas others include less than all theaforementioned items. Moreover, embodiments of the present invention arenot limited to these items, but may include additional items as will beevident to one of skill in the art. Moreover, in some embodiments,TOPAZ™ chips, available from the present assignee are utilized, althoughthis is not required by the present invention. In some embodiments,other microfluidic devices are utilized as part of a proteincrystallization process, or other microfluidic process.

Merely by way of example, another embodiment of the Automated Systemprovided according to the methods and systems of the present inventioncomprises the aforementioned elements along with items from a liquidhandling automation system. Some purposes of the liquid handlingautomation system are to automate the process for moving the sample,reagents, or stock solution from storage (chemical hotels) to thedispensing robot; creating a new reagent set from stock solutions;transferring reagents from reagent tubes to at least one microtiterplate; and dispensing the sample and reagent to the microfluidic chip.In some embodiments, the liquid handling automation system is providedby a third party. Elements of the liquid handling automation systeminclude:

Reagent and Sample Dispensing Robot Reagent and sample hotels RobotReagent and sample database (generally provided by a third party)

Moreover, in yet other embodiments of the present invention, theAutomated System, for example, an Automated TOPAZ™ System, comprises theaforementioned elements (including the liquid handling automationsystem) along with items from a Backend automation system. Some purposesfor the backend automation system are to extract the physical data (suchas crystals) from the microfluidic chips and place the completed chipinto storage or trash bins. Embodiments of the backend automation systeminclude:

Microfluidic Chip Backend Software Controller Robotics for sampleextraction from microfluidic chips

Now that a general method and system have been described, certaininformation is provided and utilized to perform, for example, proteincrystallization experiments according to embodiments of the presentinvention. As an example, the following information hierarchy includinga variety of fixed and variable parameters is utilized in a specificembodiment of the present invention. Such fixed and variable parameterswill assist the reader to understand the various embodiments containedherein. Of course, these parameters are merely examples and should notunduly limit the scope of the claims herein. One of ordinary skill inthe art would attach the broadest meaning to such parameters usingordinary meaning, although certain variations may be used depending uponthe embodiment of the present invention.

In some embodiments, this information is provided in an automated systemsuch as the ones illustrated in FIGS. 2 through 2E. As an example, theinformation would be stored in a database such as, for example, theDatabase 250 as also illustrated in FIG. 2. Accordingly, it isaccessible by the Server 252, a database management process in someembodiments, which is coupled to various system hardware and softwareelements, for example, the Experiment Manger, the FIDX, the AIX, and theCrystal Vision software. Further details of each of the informationcategories utilized in embodiments of the present microfluidic systemare described throughout the present specification and more particularlybelow. As noted below, such categories are not intended to unduly limitthe scope of the claims herein.

Information Hierarchy 1) Reagent Plate  a) Reagent Set   i) Reagent   (1) Component Formulation     (a) Component 2) Dispense Mapping 3)Sample  a) Construct Formulation   i) Construct    (1) Target  b) LigandFormulation 4) Ligand  a) Cofactor Formulation   i) Cofactor  b)Component Formulation   i) Component 5) Chip/Plate Run  a) Sample  b)Reagent Set  c) Reagent Dispense Mapping  d) Scan Session   i) DiffusionExperiment Result 6) Program Settings 7) Users

Although the general parameters have been described above, suchparameters of the information hierarchy can be further sub-divided asexplained below. The following parameter definitions and elements arenot intended to limit the claimed invention, but to merely providesexamples of various parameters utilized in embodiments of the presentinvention.

As an example, in some embodiments, a reagent set contains the designinformation for a set of reagents contained in a well plate.

The attributes of the reagent set according to an embodiment include:

Name Part Number Vendor Description Plate Format (e.g., a 96 well plateformat in a specific embodiment). List of the Reagents

Moreover, generally a reagent plate is used to track the physicalreagent user purchased from a vendor or made internally. The reagentplate according to an embodiment may include:

Barcode ID Lot number Made on Description

The reagent contains the design information for a reagent. Itsattributes according to an embodiment include:

Name Part Number Vendor Description pH Conductivity Osmolality Componentand its formulation

The dispense mapping describes how the reagents in a reagent plate aredispensed (mapped) to a chip run plate. The dispense mapping accordingto an embodiment includes:

Name Description Reagent Plate Format (i.e., source) Chip Run PlateFormat (i.e., destination) Dispensing Tip format (e.g., 1, 2, 4, or 8tips) Mapping between wells in reagent plate and chip run plate. In someembodiments, one reagent well is mapped to multiple chip run wells.

In specific embodiments, the sample, construct formulation, construct,target ligand and component are specified. Generally, the constructformulation describes a construct formula in a sample. Additionally, theligand formulation typically describes a ligand formula in a sample.Moreover, the cofactor formulation usually describes a cofactor formulain a sample. For example, these items are specified according to anembodiment by the following attributes:

Sample  Name  Barcode ID or identifier  Vendor (i.e., who produced thesample)  Part Number  Description  A list of constructs and theirformulation  A list of ligands and their formulation  A list ofcomponents and their formulation. Construct Formulation  Construct Name Concentration  Concentration Unit Construct  Construct Name  TargetName  Reference ID  Sequence  Description  URL Target  Name  ReferenceID  Sequence  Description  URL Ligand Formulation and Ligand  Ligand Concentration  Concentration Unit Ligand  Name  Reference ID Description Cofactor Formulation  Cofactor  Concentration Concentration Unit Cofactor  Name  Reference ID  Description

In other embodiments, the sample and reagent components are specified.The component formulation is the component formula in a reagent. Theseitems according to an embodiment include the following attributes.

Component Formulation  Component  Component Type  Concentration Concentration Unit  pH Component  Name  Reference ID  Description Alias Component Stock Solution  Part Number  Vendor  Component Name Concentration  Concentration Unit  pH

Additionally, information related to a chip run, in which proteincrystallization processes are performed using microfluidic chipsaccording to an embodiment of the present invention, the scan session,in which multiple images are acquired and processed to analyze one ormore chip runs according to an embodiment of the present invention, andresults, such as diffusion experiment results, are provided byembodiments of the present invention. Of course, there can be otherprocesses used according to embodiments of the present invention.According to some embodiments of the present invention, the chip run,scan session, and diffusion experiment results, according to someembodiments, include the following attributes.

An example of an overall chip run according to a specific embodiment isprovided below:

Chip Run  Chip run name (also used as Chip run template name)  Chip Type(e.g., TOPAZ ™ 1.96, 4.96, 8.96, X-ray chip or 96  microtiter plate) Crystallization Method (e.g., TOPAZ ™, Sitting Drop, Hanging Drop, Microbatch and Other)  Chip barcode  Experiment Parameters   IncubationTemperature   Hydration Level   FID Time  Sample and Reagent   Sample(s)  Reagent Set   Reagent Dispensing Mapping  Scan Sessions and Times  Destination file location   Number of scans and time (Design)   Numberof scans and time (Actual)   Start Time (Ideally, it is the time at thestart of FID).   Duration (from Start to the last image scan)  Loadingand FID   FID System ID for Loading   FID Bay ID for Loading   Loaded by(User ID)   FID System ID for FID   FID Bay ID for FID   FID by (UserID)   FID Software Version  User History   Owner of the Chip Run  Annotated by   Approved by  Status (one of the following)  Ready-to-Run    Active    Completion   Description  Scan Session  Scan Time   Scan by (User ID)   Imaging System ID   Image SystemSoftware Version   Status. One of the following:    Scan Completed   Auto Classified   Auto Processor Version  Diffusion Experiment (DE)Result   DE Number   Reagent Inlet   Sample Inlet   Auto Ranking Score  Auto Classification ID   User Classification ID   User Comment   ImageFile URL

According to embodiments of the present invention, a number of programsettings are provided. The program settings according to a specificembodiment can be systems settings, display formats, and the like. As anexample, the Database contains a list of user classification with thefollowing attributes. In a particular embodiment, these attributes arethe same as the ones defined in Crystal Vision. Moreover, in someembodiments, the user classification (annotation) settings providesettings for how to create data reports and other operations. Theprogram settings according to an embodiment of the present inventioninclude the following:

Classification Name Classification Type (Crystal, Ranked or Other) UserRanking Color Hot Key

According to other embodiments, user information is provided and stored.The user information includes parameters that uniquely and specificallyidentifies a particular user of the present methods and systemsaccording to specific embodiments. As an example, the Database generallycontains a list of users with the following attributes according to anembodiment of the present invention:

User Full Name User Description User Login Name User Password AccessRight. In some embodiments, only full access rights are supported.Status (either active or inactive)

FIG. 7 is a simplified flowchart illustrating a method of designing anexperiment according to an embodiment of the present invention. In theembodiment illustrated in FIG. 7, a method for designing a proteincrystallization experiment to screen protein samples is outlined.Although certain details of the method are also provided according tothe flow diagram illustrated by FIG. 7, this figure is not intended tounduly limit the scope of the claims herein. One of ordinary skill inthe art would recognize many variations, alternatives, andmodifications.

Select the sample to be screened (710). In some embodiments, this stepis performed by a system user. In an alternative embodiment, the sampleinformation is entered into experiment manager software if the sampleinformation has not been previously entered into the experiment managersoftware. Merely by way of example, the sample information may beentered using experiment management software such as TOPAZ™ ExperimentManager software, available from the present assignee. In someembodiments, the information entered into the experiment managementsoftware is sample tracking information, such as sample name, source,barcode identifier, and the like. Additionally, in some embodiments, theprotein construct and concentration, the co-crystallization componentsand concentrations, and sample buffer components and concentrations areentered.

Select the screening reagents to be used for screening (715). In someembodiments, this step is performed by a system user. In a particularembodiment, the screening reagent information is entered into theexperiment manager software if the screening reagent information has notbeen previously entered into the experiment manager software. Merely byway of example, the screening reagent information may be entered usingexperiment management software such as TOPAZ™ Experiment Managersoftware, available from the present assignee. In some embodiments, theinformation entered into the experiment management software includesreagent tracking information, such as reagent name, source, type ofmicrofluidic plate, barcode identifier, and the like. In a particularembodiment, the type of microfluidic plate is a microtiter plate (e.g.,a 96 well plate). Additionally, in some embodiments, reagent formationinformation for each well (e.g., salt, precipitant, buffer and the like)is also entered into the experiment manager software.

Select a type of microfluidic device or chip (720). In some embodiments,this step is performed by a system user. In a specific embodiment,TOPAZ™ chips, available from the present assignee are selected. Merelyby way of example, a TOPAZ™ 1.96 chip or a TOPAZ™ 4.96 chip, providingformats that screen one or four protein samples, respectively, against96 reagents, are selected in a particular embodiment of the presentinvention.

Select a dispensing scheme (725). In some embodiments, this step isperformed by a system user. Generally, the dispensing scheme includes amapping from a screening reagent plate to the microfluidic chip, forexample, the TOPAZ™ chip.

Select a suitable workflow template, which defines the sequence of jobsand timing that is to be performed on the microfluidic chip (730). Insome embodiments, this step is performed by a system user. In someembodiments, the workflow template is defined, as discussed in relationto FIG. 12 if the suitable workflow template does not already exist.

Select an owner for the protein crystallization experiment (735). Insome embodiments, this step is performed by a system user. Moreover, inan embodiment, the owner is, for example, a technician.

Save the experimental design information a database (740). In aparticular embodiment, the database is a database provided as part of aTOPAZ™ System available from the present assignee.

Inform the owner of the protein crystallization experiment that thescreening experiment is ready to run (745). In some embodiments, aworkflow manager is utilized to inform the owner. In specificembodiments of the present invention, the owner is notified by e-mail,pager, SMS, instant messenger (IM), voice mail, and the like.

Run or perform the protein crystallization experiment (750). Additionaldetails regarding the running of the experiment are provided below inrelation to the discussion of FIG. 8.

Perform other steps, as desired (755).

It should be appreciated that the specific steps illustrated aboveprovide a particular method of designing a process (sometimes anexperimental process) according to an embodiment of the presentinvention. Other sequence of steps may also be performed according toalternative embodiments. For example, alternative embodiments of thepresent invention may perform the steps outlined above in a differentorder. Moreover, the individual steps illustrated by this method mayinclude multiple sub-steps that may be performed in various sequences asappropriate to the individual step. Furthermore, additional steps may beadded or removed depending on the particular applications. One ofordinary skill in the art would recognize many variations,modifications, and alternatives. Further details of methods andresulting structures of the present microfluidic system have beendescribed throughout the present specification and more particularlybelow.

FIG. 8 is a simplified flowchart illustrating a method of running anexperiment according to an embodiment of the present invention.

Prepare a microfluidic chip (810). Merely by way of example, in someembodiments, the microfluidic chip is a TOPAZ™ chip, for example, aTOPAZ™ 1.96 chip or a TOPAZ™ 4.96 chip, providing formats that screenone or four protein samples, respectively, against 96 reagents.Moreover, in an embodiment this step is performed by a system user. In aspecific embodiment, the microfluidic chip is prepared according to aworkflow protocol defined during the experimental design process, asdiscussed in relation to FIG. 7.

Enter the barcode of the microfluidic chip, into a database andassociate the chip with a defined experiment or process (815). In someembodiments, the microfluidic chip is a TOPAZ™ chip and this step isperformed by a system user. Moreover, in other embodiments, theexperiment is defined as illustrated in relation to FIG. 7.

Place the microfluidic chip into a Liquid Handling System (820). In someembodiments, this step is performed by a system user. Liquid HandlingSystems are described in additional detail throughout the presentspecification.

Scan the barcode of the microfluidic device (825). In some embodiments,this step is performed automatically by the Liquid Handling System usingappropriate hardware and software.

Make a query to the Workflow Manager, the query including the chipbarcode (830). In some embodiments, the Workflow Manager is a TOPAZ™Workflow Manager. Moreover, in other embodiments, this step is performedby the Liquid Handling System through a standard software communicationprotocol, such as SOAP (Simple Object Access Protocol)).

Return the job request from the Workflow Manager to the Liquid HandlingSystem (835). In a particular embodiment, the job request instructs theLiquid Handling System with respect to what samples and reagents arerequired and how to dispense them into the microfluidic chip, forexample a TOPAZ™ Chip.

Dispense the sample and reagent into the microfluidic chip according toinstructions provided with the job request (840). In some embodiments,this step is performed automatically by the Liquid Handling System usingappropriate hardware and software.

Place the microfluidic chip in an Automation Input Stack after thecompletion of the dispense step (845). In some embodiments, this step isperformed automatically by the Liquid Handling System using appropriatehardware and software.

Detect the presence of the microfluidic chip in the Automation system(850). In some embodiments, this step is performed automatically by theAutomation system by a process of scanning the barcode associated withthe microfluidic device.

Instruct the transfer robot to transfer the microfluidic chip to a freeinterface diffusion (FID) crystallizer for loading of the protein sampleand reagent into the microfluidic device (855). In a specificembodiment, the FID crystallizer is a TOPAZ™ FID crystallizer (FIDX)available from the present assignee. Moreover, in some embodiments, thisstep is performed by a Workflow Manager.

Instruct the transfer robot to transfer the microfluidic chip to aninspection station to perform a baseline scan, sometimes referred to asa time t₀ scan (860). Baseline scans are performed at various stagesdepending on the particular application. As one of skill in the art willappreciate, acquiring a baseline image enables techniques to reducebackground noise, among other reasons. For example, in a specificembodiment, the time t₀ scan is acquired before an experiment is begunby loading the reagent and sample into the wells on the chip but notopening the valves to allow for diffusion of the reagents and samples.In this specific embodiment, the image is acquired before the FIDprocess begins. Of course, other embodiments acquire the baseline scanat other stages depending on the particular application. In someembodiments, the instructions are provided by the Workflow Manager. In aspecific embodiment, the inspection station is a TOPAZ™ AUTOINSPEXT™Workstation (AIX), available from the present assignee. In someembodiments, this step is an optional step as baseline scans or imagesare not utilized.

Instruct the transfer robot to transfer the microfluidic chip to the FIDcrystallizer to initiate the protein crystallization process (865). In aspecific embodiment, the FID crystallizer is the TOPAZ™ FIDX and valvespresent on a TOPAZ™ chip are controlled to release the sample and thereagent and initiate the FID process. As one of skill in the art willappreciate, in some cases, the FID process results in the formation of acrystal in the well region of the microfluidic chip. In the presentspecification, references to a well region or well regions include, butare not limited to chambers, micro-chambers, channels, micro-channels,reaction chambers, and the like.

Transfer the microfluidic chip to a microfluidic chip hotel (870). Insome embodiments, the instructions are provided by the workflow manager.Depending on the length of time designed for the FID process, along withthe status of other tasks performed by the protein crystallizationexperiment system, it may be desirable to store the microfluidic chipsin a chip hotel. The chip hotel provides control of the temperature andhumidity environment surrounding the microfluidic chip, among otherparameters. In some embodiments, this is an optical step as themicrofluidic chip remains in the FID crystallizer during the duration ofthe FID process. In some embodiments, the system will transfer themicrofluidic chip to the FID crystallizer to terminate the FID process.According to an embodiment of the present invention, in process flows inwhich the microfluidic device is stored in the microfluidic chip hotel,the workflow manager instructs the transfer robot to transfer themicrofluidic chip to the FID crystallizer. In a specific embodiment, theFID crystallizer is the TOPAZ™ FIDX and valves present on a TOPAZ™ chipare controlled to load and manipulate the sample and the reagent for theprotein crystallization processes, and also to terminate the FIDprocess.

Instruct the transfer robot to transfer the microfluidic chip to theinspection system to perform scans as defined by the workflow protocol(875). In a specific embodiment, the inspection station is a TOPAZ™AUTOINSPEX™ Workstation (AIX), available from the present assignee and anumber of images are acquired and processed at time intervals defined bythe workflow protocol. In other embodiments, results from initialmeasurements made using the inspection system are analyzed to determinethe time intervals for subsequent image acquisition and/or processing.

Automatically process at least one of the number of images acquiredusing the inspection system to determine a parameter, for example, acrystal ranking, associated with the protein crystallization process(880). In some embodiments, one or more parameters are determined andstored in a database, thereby updating the database. In a particularembodiment, the database is a database provided as part of the TOPAZ™System, for example, a TOPAZ™ database.

Provide the system user with information related to the proteincrystallization experiment (885). In some embodiments, the system useris provided with this information by a server coupled to the inspectionsystem, for example, a TOPAZ™ database server. Moreover, in a specificembodiment, the information includes the crystal ranking determined inthe previous step.

Access a software package to view the results of the proteincrystallization experiment and annotate the experimental results ifdesired (890). In some embodiments, the system user uses the TOPAZ™ AIXSoftware suite, including Crystal Vision software to view theexperimental results and further annotate the experiments as desired.

Perform other steps as necessary (895).

According to embodiments of the present invention, after performing theaforementioned steps, the system user may pursue a variety of optionsaccording to this method. For example, the user may desire to continuethe protein crystallization experiment, acquiring and processingadditional images of portions of the microfluidic chip. As will beevident to one of skill in the art, the comparison of images acquired asa function of time may yield additional information related to theprotein crystallization process. Additionally, the user may select anoption in which another screening process is initiated, using theworkflow protocol defined separately. Moreover, based on the informationcommunicated in step 885, the user may desire to perform an optimizationexperiment. Furthermore, the user may select to perform a translationexperiment, utilizing a microfluidic diffraction chip such as the TOPAZ™Chip, a classic method, (e.g. Sitting Drop, Hanging Drop, andMicrobatch), and the like.

It should be appreciated that the specific steps illustrated in steps 1through 14 provide a particular method of running an experimentaccording to an embodiment of the present invention. Other sequence ofsteps may also be performed according to alternative embodiments. Forexample, alternative embodiments of the present invention may performthe steps outlined above in a different order. Moreover, the individualsteps illustrated by this method may include multiple sub-steps that maybe performed in various sequences as appropriate to the individual step.For example, the step of acquiring and processing an image may containsub-steps, such as the alignment and focusing of optical elements.Furthermore, additional steps may be added or removed depending on theparticular applications. One of ordinary skill in the art wouldrecognize many variations, modifications, and alternatives. Furtherdetails of methods and resulting structures of the present microfluidicsystem have been described throughout the present specification and moreparticularly below.

FIG. 9 is a simplified flowchart illustrating a method of optimizing anexperiment according to an embodiment of the present invention.

Select a set of conditions for optimization (910). In some embodimentsof the present invention, this step is performed by a system user. In anembodiment of the present invention, an experiment manager, for examplea TOPAZ™ Experiment Manager creates a new reagent set using standardexperiment methodology.

Save the new reagent set using the experiment manager, for example, theTOPAZ™ Experiment Manager to the Database (915). In an embodiment, thereagent set is saved in one or more memories of a computing system.Next, place a job request with a Workflow Manager. In a specificembodiment, the Workflow Manager is a TOPAZ™ Workflow Manager.

Post the job request with the reagent formulation to the Liquid HandlingSystem (920). In some embodiments, this step is performed by theWorkflow Manager.

Make new sets according to the formulation provided in step 910 (925).In some embodiments, this step is performed automatically by the LiquidHandling System using appropriate hardware and software.

Place the created reagent sets in the reagent hotels (930). In someembodiments, this step is performed automatically by the Liquid HandlingSystem using appropriate hardware and software.

Design a new experiment with the newly created optimization reagent andthe required sample (935). In some embodiments, this step is performedin parallel with the reagent preparation and delivery steps, but this isnot required by the present invention. In some embodiments of thepresent invention, this step is performed by a system user.

Select a type of microfluidic device or chip (940). In some embodiments,this step is performed by a system user. In a specific embodiment,TOPAZ™ chips, available from the present assignee are selected. Merelyby way of example, a TOPAZ™ 1.96 chip or a TOPAZ™ 4.96 chip, providingformats that screen one or four protein samples, respectively, against96 reagents, are selected in a particular embodiment of the presentinvention.

Select a dispensing scheme (945). In some embodiments, this step isperformed by a system user. Generally, the dispensing scheme includes amapping from an optimization reagent plate to the microfluidic chip, forexample, the TOPAZ™ chip.

Select a suitable workflow template, which defines the sequence of jobsand timing that is to be performed on the microfluidic chip (950). Insome embodiments, this step is performed by a system user. In someembodiments, the workflow template is defined, as discussed in relationto FIG. 12 if the suitable workflow template does not already exist.

Select an owner for the protein crystallization optimization experiment(955). In some embodiments, this step is performed by a system user.Moreover, in an embodiment, the owner is, for example, a technician.

Save the experimental design information a database including one ormore memories of a computing system (960). In a particular embodiment,the database is a database provided as part of a TOPAZ™ System availablefrom the present assignee.

Inform the owner of the protein crystallization optimization experimentthat the optimization experiment is ready to run (965). In someembodiments, a workflow manager is utilized to inform the owner. Inspecific embodiments of the present invention, the owner is notified bye-mail, pager, SMS, instant messenger (IM), voice mail, and the like.

Run or perform the protein crystallization optimization experiment(970). Additional details regarding the running of the optimizationexperiment are provided above in relation to the discussion of FIG. 8.

Perform other steps, as desired (975).

FIG. 10 is a simplified flowchart illustrating a method of translatingan experiment according to an embodiment of the present invention.

Select a set of conditions for translation. In some embodiments of thepresent invention, this step is performed by a system user. In anembodiment of the present invention, an experiment manager, for examplean Experiment Manager creates a new reagent set using a standardtranslation protocol or a TOPAZ™ specific translation protocol. Merelyby way of example, in a specific embodiment, the Experiment Manager is aTOPAZ™ Experiment Manager.

Save the new reagent set using the experiment manager, for example, theTOPAZ™ Experiment Manager to a Database (1015). In an embodiment, thereagent set is saved in one or more memories of a computing system.Next, place a job request with a Workflow Manager. In a specificembodiment, the Workflow Manager is a TOPAZ™ Workflow Manager.

Post the job request with the reagent formulation to the Liquid HandlingSystem (1020). In some embodiments, this step is performed by theWorkflow Manager.

Make new sets according to the formulation provided in step 1010 (1025).In some embodiments, this step is performed automatically by the LiquidHandling System using appropriate hardware and software.

Place the created reagent sets in the reagent hotels (1030). In someembodiments, this step is performed automatically by the Liquid HandlingSystem using appropriate hardware and software.

Design a new experiment with the newly created optimization reagent andthe required sample (1035). In some embodiments, this step is performedin parallel with the reagent preparation and delivery steps, but this isnot required by the present invention. In some embodiments of thepresent invention, this step is performed by a system user.

Select a type of microfluidic device or chip (1040). In someembodiments, this step is performed by a system user. In a specificembodiment, TOPAZ™ chips, available from the present assignee areselected. Merely by way of example, a TOPAZ™ 1.96 chip or a TOPAZ™ 4.96chip, providing formats that screen one or four protein samples,respectively, against 96 reagents, are selected in a particularembodiment of the present invention.

Select a dispensing scheme (1045). In some embodiments, this step isperformed by a system user. Generally, the dispensing scheme includes amapping from an optimization reagent plate to the microfluidic chip, forexample, the TOPAZ™ chip.

Select a suitable workflow template, which defines the sequence of jobsand timing that is to be performed on the microfluidic chip (1050). Insome embodiments, this step is performed by a system user. In someembodiments, the workflow template is defined, as discussed in relationto FIG. 12 if the suitable workflow template does not already exist.

Select an owner for the protein crystallization optimization experiment(1055). In some embodiments, this step is performed by a system user.Moreover, in an embodiment, the owner is, for example, a technician.

Save the experimental design information a database including one ormore memories of a computing system (1060). In a particular embodiment,the database is a database provided as part of a TOPAZ™ System availablefrom the present assignee.

Inform the owner of the protein crystallization optimization experimentthat the optimization experiment is ready to run (1065). In someembodiments, a workflow manager is utilized to inform the owner. Inspecific embodiments of the present invention, the owner is notified bye-mail, pager, SMS, instant messenger (IM), voice mail, and the like.

Run or perform the protein crystallization optimization experiment(1070). Additional details regarding the running of the translationexperiment are provided above in relation to the discussion of FIG. 8.

Perform other steps, as desired (1075).

FIG. 11 is a simplified flowchart illustrating a method of translationoptimization for an experiment according to an embodiment of the presentinvention.

Select a set of conditions for translation optimization (1110). In someembodiments of the present invention, this step is performed by a systemuser. In an embodiment of the present invention, an experiment manager,for example an Experiment Manager creates a new reagent set using astandard translation optimization protocol. Merely by way of example, ina specific embodiment, the Experiment Manager is a TOPAZ™ ExperimentManager.

Save the new reagent set using the experiment manager, for example, theTOPAZ™ Experiment Manager to a Database (1115). In an embodiment, thereagent set is saved in one or more memories of a computing system.Next, place a job request with a Workflow Manager. In a specificembodiment, the Workflow Manager is a TOPAZ™ Workflow Manager.

Post the job request with the reagent formulation to the Liquid HandlingSystem (1120). In some embodiments, this step is performed by theWorkflow Manager.

Make new sets according to the formulation provided in step 1110 (1125).In some embodiments, this step is performed automatically by the LiquidHandling System using appropriate hardware and software.

Place the created reagent sets in the reagent hotels (1130). In someembodiments, this step is performed automatically by the Liquid HandlingSystem using appropriate hardware and software.

Design a new experiment with the newly created optimization reagent andthe required sample (1135). In some embodiments, this step is performedin parallel with the reagent preparation and delivery steps, but this isnot required by the present invention. In some embodiments of thepresent invention, this step is performed by a system user.

Select a type of microfluidic device or chip (1140). In someembodiments, this step is performed by a system user. In a specificembodiment, TOPAZ™ chips, available from the present assignee areselected. Merely by way of example, a TOPAZ™ 1.96 chip or a TOPAZ™ 4.96chip, providing formats that screen one or four protein samples,respectively, against 96 reagents, are selected in a particularembodiment of the present invention.

Select a dispensing scheme (1145). In some embodiments, this step isperformed by a system user. Generally, the dispensing scheme includes amapping from an optimization reagent plate to the microfluidic chip, forexample, the TOPAZ™ chip.

Select a suitable workflow template, which defines the sequence of jobsand timing that is to be performed on the microfluidic chip (1150). Insome embodiments, this step is performed by a system user. In someembodiments, the workflow template is defined, as discussed in relationto FIG. 12 if the suitable workflow template does not already exist.

Select an owner for the protein crystallization optimization experiment(1155). In some embodiments, this step is performed by a system user.Moreover, in an embodiment, the owner is, for example, a technician.

Save the experimental design information a database including one ormore memories of a computing system (1160). In a particular embodiment,the database is a database provided as part of a TOPAZ™ System availablefrom the present assignee.

Inform the owner of the protein crystallization optimization experimentthat the optimization experiment is ready to run (1165). In someembodiments, a workflow manager is utilized to inform the owner. Inspecific embodiments of the present invention, the owner is notified bye-mail, pager, SMS, instant messenger (IM), voice mail, and the like.

Run or perform the protein crystallization translation optimizationexperiment (1170). Additional details regarding the running of thetranslation optimization experiment are provided above in relation tothe discussion of FIG. 8.

Perform other steps, as desired (1175).

FIG. 12 is a simplified flowchart illustrating a workflow templateaccording to an embodiment of the present invention.

Select a level of hydration for the microfluidic device (1210).

Select a loading protocol for loading the reagent and sample into themicrofluidic device or chip (1215). Generally, the loading protocol willinclude information As one of skill in the art will appreciate, in someembodiments of the present invention, this step is dependent on the typeof microfluidic device selected for the protein crystallizationexperiment.

Select an active FID protocol (1220). Typically, the active FID protocolincludes a length of time during which the interface line is open, amongother variables.

Determine whether a baseline (time t₀) image is to be acquired (1225).In some embodiments, no baseline image is acquired and thisdetermination is made in this step.

Select the number of images to be acquired and the timing with whichthis number of images is to be acquired (1210).

Select other parameters as desired (1230).

According to embodiments of the present invention, there are multipleexamples of protein crystallization experiments that are run utilizingthe Database Application Suite. Some of these experiments involvemultiple software components, while others will only involve onecomponent. One of ordinary skill in the art would recognize manyvariations, modifications, and alternatives. Additionally, some of theseexamples of using methods and systems according to embodiments of thepresent invention do not constitute performance of entire experiments,but merely portions or sub-elements thereof.

As a first example, a method for forming a reagent set according to anembodiment of the present invention is provided by the following processflow. As described more fully below, a reagent set formed according toembodiments of the present invention may be used in performing proteincrystallization experiments as will be evident to one of skill in theart. FIG. 13 is a simplified flowchart illustrating operations performedaccording to an exemplary embodiment of the present invention.Accordingly, the reference numerals in the following process flow referto FIG. 13.

-   -   1310. Start Experiment Manager Software if desired on an        automated system, which has been described herein.    -   1315. Create the required reagent set by importing the reagent        set using for example, a comma separated values (CSV) file,        provided by a first entity. In some embodiments of the present        invention, the first entity is Fluidigm Corporation        (“Fluidigm”).    -   1320. Edit the reagent set information, if desired, including        adding or removing and/or modifying any of the reagents in the        reagent set, to form a desired reagent set by a second entity or        user of the edited reagent set.    -   1325. Save the created reagent set into one or more memories of        a computing system.    -   1330. Perform any other steps, as desired.

As shown, above, the above steps can be used to form a reagent setaccording to an embodiment of the present invention. Other alternativescan also be provided where steps are added, one or more steps areremoved, or one or more steps are provided in a different sequencewithout departing from the scope of the claims herein. In certainembodiments, any of these steps may be combined with the others recitedherein.

As a second example, a method for forming a new non-Fluidigm reagent setaccording to an embodiment of the present invention is provided by thefollowing process flow. FIG. 14 is a simplified flowchart illustratingoperations performed according to an exemplary embodiment of the presentinvention. Accordingly, the reference numerals in the following processflow refer to FIG. 14.

-   -   1410. Start Microsoft Excel software on an automated system,        which has been described herein.    -   1415. Open the reagent set using for example, a CSV file        template provided by a first entity. In some embodiments, the        first entity is Fluidigm.    -   1420. Open the reagent data set provided by a third party vendor        or a reagent data set created, for example, internally.    -   1425. Copy and paste the desired reagent data information to the        CSV file template provided by the first entity.    -   1430. When finished with the copy and paste operation, save the        reagent data information to a CSV file stored in one or more        memories of a computing system.    -   1435. Start Experiment Manager Software if desired on an        automated system, which has been described herein.    -   1440. Create the required reagent set by importing the newly        created CSV file.    -   1445. Edit the reagent set information, if desired, including        adding or removing and/or modifying any of the reagents in the        reagent set, to form a desired reagent set by a second entity or        user of the edited reagent set.    -   1450. Save the created reagent set into one or more memories of        a computing system.    -   1455. Perform any other steps, as desired.

As shown, above, the above steps can be used to form a non-Fluidigmreagent set according to an embodiment of the present invention. Otheralternatives can also be provided where steps are added, one or moresteps are removed, or one or more steps are provided in a differentsequence without departing from the scope of the claims herein. Incertain embodiments, any of these steps may be combined with the othersrecited herein.

In a third example, a method of adding a physical reagent plateaccording to an embodiment of the present invention is provided by thefollowing process flow. In general, a precondition for this example isthat the reagent set (i.e., design information for the reagent set) forthe physical reagent plate is already stored in the database. FIG. 15 isa simplified flowchart illustrating operations performed according to anexemplary embodiment of the present invention. Accordingly, thereference numerals in the following process flow refer to FIG. 15.

-   -   1510. Start Experiment Manager Software if desired on an        automated system, which has been described herein.    -   1520. Select the reagent set for the physical reagent plate.    -   1530. Create a new entry in the physical reagent plate list.    -   1540. Enter the barcode identifier, batch number, and other        physical information for the new entry and then save the new        entry into one or more memories of a computing system.    -   1550. Perform any other steps, as desired.

As shown, above, the above steps can be used to add a physical reagentplate according to an embodiment of the present invention. Otheralternatives can also be provided where steps are added, one or moresteps are removed, or one or more steps are provided in a differentsequence without departing from the scope of the claims herein. Incertain embodiments, any of these steps may be combined with the othersrecited herein.

As a fourth example, a method for forming a new protein target accordingto an embodiment of the present invention is provided by the followingprocess flow. FIG. 16 is a simplified flowchart illustrating operationsperformed according to an exemplary embodiment of the present invention.Accordingly, the reference numerals in the following process flow referto FIG. 16.

-   -   1610. Start Experiment Manager Software if desired on an        automated system, which has been described herein.    -   1620. Create a new protein target.    -   1630. Enter the required attributes for the target.    -   1640. Save the new entry into one or more memories of a        computing system.    -   1650. Perform any other steps, as desired.

As shown, above, the above steps can be used to form a new proteintarget according to an embodiment of the present invention. Otheralternatives can also be provided where steps are added, one or moresteps are removed, or one or more steps are provided in a differentsequence without departing from the scope of the claims herein. Incertain embodiments, any of these steps may be combined with the othersrecited herein.

As a fifth example, a method of forming a new protein constructaccording to an embodiment of the present invention is provided by thefollowing process flow. Generally, a precondition for this process isthat the protein target for the new construct is already created andstored in the Database. Merely by way of example, the method of forminga new protein target discussed in relation to the fourth example is usedin a specific embodiment of the present invention. Typically, there is adefault construct (full length) for each protein target. FIG. 17 is asimplified flowchart illustrating operations performed according to anexemplary embodiment of the present invention. Accordingly, thereference numerals in the following process flow refer to FIG. 17.

-   -   1710. Start Experiment Manager Software if desired on an        automated system, which has been described herein.    -   1720. Select the protein target.    -   1730. Create a new entry in the construct list.    -   1740. Enter the new construct information for the selected        protein target.    -   1750. Save the new entry into one or more memories of a        computing system.    -   1760. Perform any other steps, as desired.

As shown, above, the above steps can be used to form a new proteinconstruct according to an embodiment of the present invention. Otheralternatives can also be provided where steps are added, one or moresteps are removed, or one or more steps are provided in a differentsequence without departing from the scope of the claims herein. Incertain embodiments, any of these steps may be combined with the othersrecited herein.

In a sixth example, a method of forming a new ligand according to anembodiment of the present invention is provided by the following processflow. FIG. 18 is a simplified flowchart illustrating operationsperformed according to an exemplary embodiment of the present invention.Accordingly, the reference numerals in the following process flow referto FIG. 18.

-   -   1810. Start Experiment Manager Software if desired on an        automated system, which has been described herein.    -   1820. Select the ligand.    -   1830. Create a new ligand.    -   1840. Enter the new ligand information.    -   1850. Save the new entry into one or more memories of a        computing system.    -   1860. Perform any other steps, as desired.

As shown, above, the above steps can be used to form a new ligandaccording to an embodiment of the present invention. Other alternativescan also be provided where steps are added, one or more steps areremoved, or one or more steps are provided in a different sequencewithout departing from the scope of the claims herein. In certainembodiments, any of these steps may be combined with the others recitedherein.

In a seventh example, a method of forming a new protein sample accordingto an embodiment of the present invention is provided by the followingprocess flow. Typically, the protein target(s), construct(s), ligand(s),and buffer(s) for the new protein sample are already created and storedin the Database. FIG. 19 is a simplified flowchart illustratingoperations performed according to an exemplary embodiment of the presentinvention. Accordingly, the reference numerals in the following processflow refer to FIG. 19.

-   -   1910. Start Experiment Manager Software if desired on an        automated system, which has been described herein.    -   1920. Create a new entry in the protein sample list.    -   1930. Select the protein target(s).    -   1940. Select the construct(s) for the sample.    -   1950. Select the ligand(s), concentration(s), and unit(s) in the        case of co-crystallization.    -   1960. Select the component(s), concentration(s), unit(s), and        pH(s) for the chemical components.    -   1970. Define the other desired attributes of the new protein        sample.    -   1980. Save the new entry into one or more memories of a        computing system.    -   1990. Perform any other steps, as desired.

As shown, above, the above steps can be used to form a new proteinsample according to an embodiment of the present invention. Otheralternatives can also be provided where steps are added, one or moresteps are removed, or one or more steps are provided in a differentsequence without departing from the scope of the claims herein. Incertain embodiments, any of these steps may be combined with the othersrecited herein.

In an eighth example, a method of forming a new dispensing mappingaccording to an embodiment of the present invention is provided by thefollowing process flow. FIG. 20 is a simplified flowchart illustratingoperations performed according to an exemplary embodiment of the presentinvention. Accordingly, the reference numerals in the following processflow refer to FIG. 20.

-   -   2010. Start Experiment Manager Software if desired on an        automated system, which has been described herein.    -   2020. Create a new entry in the dispensing list.    -   2030. Define the name of the dispensing mapping.    -   2040. Select the reagent plate format and chip run plate format.    -   2050. Select the dispensing tip format (e.g., 1, 2, 4, or 8        tips).    -   2060. Define the well-to-well relationship between the reagent        plate and chip run plate.    -   2070. Save the new entry into one or more memories of a        computing system.    -   2080. Perform any other steps, as desired.

As shown, above, the above steps can be used to form a new dispensingmapping according to an embodiment of the present invention. Otheralternatives can also be provided where steps are added, one or moresteps are removed, or one or more steps are provided in a differentsequence without departing from the scope of the claims herein. Incertain embodiments, any of these steps may be combined with the othersrecited herein.

In a ninth example, a method of forming a new chip run templateaccording to an embodiment of the present invention is provided by thefollowing process flow. Generally, a precondition for this process isthat the reagent set, protein sample(s), and the dispensing mapping arealready created and stored in the Database. FIG. 21 is a simplifiedflowchart illustrating operations performed according to an exemplaryembodiment of the present invention. Accordingly, the reference numeralsin the following process flow refer to FIG. 21.

-   -   2110. Start Experiment Manager Software if desired on an        automated system, which has been described herein.    -   2120. Define chip run template name.    -   2130. Select Chip Type. In some embodiments, this step is        optional.    -   2140. Define the chip run file location. In some embodiments,        this step is optional.    -   2150. Define the number of scans and scan time, incubation        temperature, and hydration level. In some embodiments, these        steps are optional.    -   2160. Select Reagent Set. In some embodiments, this step is        optional.    -   2170. Select dispensing mapping. In some embodiments, this step        is optional.    -   2180. Save the chip run template in the template folder into one        or more memories of a computing system.    -   2190. Perform any other steps, as desired.

As shown, above, the above steps can be used to form a new chip runtemplate according to an embodiment of the present invention. Otheralternatives can also be provided where steps are added, one or moresteps are removed, or one or more steps are provided in a differentsequence without departing from the scope of the claims herein. Incertain embodiments, any of these steps may be combined with the othersrecited herein.

In a tenth example, a method of forming a chip run in the ready-to-runlist according to an embodiment of the present invention is provided bythe following process flow. Generally, a precondition for this processis that the reagent set, protein sample(s), and the dispensing mappingare already created and stored in the Database. FIG. 22 is a simplifiedflowchart illustrating operations performed according to an exemplaryembodiment of the present invention. Accordingly, the reference numeralsin the following process flow refer to FIG. 22.

-   -   2210. Start Experiment Manager Software if desired on an        automated system, which has been described herein.    -   2215. Selection of a chip run template or none.    -   2220. Define chip run name and select the chip type.    -   2225. Define the chip run file location.    -   2230. Define the number of scans and scan time, incubation        temperature, and hydration level.    -   2235. Select sample(s) and the mapping if necessary (e.g., 4.96        or 8.96 chip)    -   2240. Select the reagent set.    -   2245. Select the dispensing mapping. Merely by way of example,        the dispensing mapping is created using the method discussed in        relation to the eighth example above.    -   2250. Print a chip run worksheet or deliver, for example,        through email or other communication means, the worksheet to the        staff member who will conduct the chip run. The worksheet will        generally be in a Hyper Text Markup Language (HTML) format.    -   2255. When finished, save the chip run to the ready-to-run        folder in one or more memories of a computing system.    -   2260. Perform any other steps, as desired.

As shown, above, the above steps can be used to form a chip run in theready-to-run list according to an embodiment of the present invention.Other alternatives can also be provided where steps are added, one ormore steps are removed, or one or more steps are provided in a differentsequence without departing from the scope of the claims herein. Incertain embodiments, any of these steps may be combined with the othersrecited herein.

In an eleventh example, a method for running a microfluidic chip runutilizing a designed chip run according to an embodiment of the presentinvention is provided by the following process flow. Generally, as aprecondition, the designed chip run is already created as discussed inrelation to the tenth example. FIG. 23 is a simplified flowchartillustrating operations performed according to an exemplary embodimentof the present invention. Accordingly, the reference numerals in thefollowing process flow refer to FIG. 23.

-   -   2310. A first user designs a set of chip runs and saves them to        the ready-to-run list in one or more memories of a computing        system (see the tenth example for creating a chip run in the        ready-to-run list).    -   2315. Send the new chip run notice to a second user through        email, phone, or the like.    -   2320. The second user starts Experiment Manager Software on an        automated system, which has been described herein. The second        user uses the Experiment Manager or Internet Explorer to find        the details of the new chip run and optionally print the work        order.    -   2325. The second user takes a chip from the stock. In some        embodiments, this step is not a TOPAZ™ database function, but is        performed off-line by a technician or other system operator.    -   2330. Hydrate the chip to the level of desired hydration level.        In some embodiments, this step is not a TOPAZ™ database        function.    -   2335. Prime the chip if desired. Generally, this is a FID        function and in some embodiments, no database information        exchange is needed.    -   2340. Dispense the reagent and sample into the chip.    -   2345. Go to the FID crystallizer and perform the loading.        Typically, at the same time, the FID software will ask the user        to:

Associate this chip to a chip run in the Ready-to-Run list.

Enter the physical information for the Sample and Reagent Plate

Additionally, typically, the FID will update the Database with systeminformation.

-   -   2350. Go to the AIX for a time t₀ scan if necessary. Since the        chip run information is already stored in the Database, the AIX        will automatically update the chip run information if a time t₀        scan is performed. The chip run is generally identified by the        barcode identifier.    -   2355. Go to the FID crystallizer and perform the FID. The FID        will update the database with FID time and system information.    -   2360. Go to the AIX system and scan the data. AIX will update        the information for this chip run in the Database.    -   2365. The data will be automatically processed by an Auto        processor in some embodiments and the results will be        automatically uploaded into the Database.    -   2370. A user utilizes a software program, for example, Crystal        Vision, to annotate the results.    -   2375. According to some embodiments, whenever a user saves the        annotation results, the Database will update the “Annotated by”        field of the chip run in one or more memories of a computing        system.    -   2380. Additionally, whenever the same user or a different user        approves the annotation results, the user will use a software        program, for example, Crystal Vision, to update the “Approved        by” field of the chip run in one or more memories of a computing        system, and make the chip run complete.    -   2385. Provide a Crystallization Result:    -   For a diffraction chip,        -   2386 a. If there is a good quality crystal:            -   Extract the crystal.            -   Go to the beam. In some embodiments, this step involves                performing crystallography including, but not limited                to, x-ray diffraction crystallography.                -   Structure resolved: The END.                -   Structure not resolved: Repeat the experiment or use                    a different reagent set design.        -   2386 b. If there is not a good quality crystal:            -   Repeat the experiment or use a different reagent set                design.        -   2386 c. If there is no crystal:            -   Select a different reagent set and re-run the                experiment.    -   For a screening chip,        -   2387 a. If there is a good quality crystal:            -   From the crystallization condition, create a new reagent                set or select a reagent set for the diffraction run.        -   2387 b. If there is not a good quality crystal:            -   From a set of the crystallization conditions, create a                new reagent set or select a reagent set for an                optimization chip run.        -   2387 c. If there is no crystal:            -   Select a different reagent set and re-run the                experiment.    -   2390. Perform any other steps, as desired.

As shown, above, the above steps can be used to run a microfluidic chiprun utilizing a designed chip run according to an embodiment of thepresent invention. Other alternatives can also be provided where stepsare added, one or more steps are removed, or one or more steps areprovided in a different sequence without departing from the scope of theclaims herein. In certain embodiments, any of these steps may becombined with the others recited herein.

As a twelfth example, a method for running a microfluidic chip runwithout a designed chip run according to an embodiment of the presentinvention is provided by the following process flow. FIG. 24 is asimplified flowchart illustrating operations performed according to anexemplary embodiment of the present invention. Accordingly, thereference numerals in the following process flow refer to FIG. 24.

-   -   2410. A second user takes a chip from the stock (generally not a        TOPAZ™ database function) and starts a chip run.    -   2415. Hydrate the chip to the level of required hydration level        if necessary (not a TOPAZ™ database function).    -   2420. Prime the chip if needed (generally a FID function, but no        database information exchange needed).    -   2425. Dispense the reagent and sample into the chip.    -   2430. Go to the FID crystallizer and perform the loading. Since        there is no chip run in the “Ready-to-Run” list, the second user        will generally have to create a new chip run on the fly in the        FID software. To create a new chip run, a user generally has to        enter the following information into one or more memories of a        computing system:

Chip Run Name Chip Type Crystallization Method Reagent Set and ReagentPlate (Optional) Dispensing Mapping (Optional) Sample (Optional)

-   -   2435. After entering the chip run information into one or more        memories of a computing system, the user will continue and        complete the loading.    -   After completion of 2435, additional steps as provided by steps        2350 to 2385 in the eleventh example are followed.    -   2480. Before the completion of the chip run, in a specific        embodiment of the present invention, the first user will use the        Experiment Manager to complete the required information for this        chip run.    -   2485. Perform any other steps, as desired.

As shown, above, the above steps can be used to run a microfluidic chiprun without a designed chip run according to an embodiment of thepresent invention. Other alternatives can also be provided where stepsare added, one or more steps are removed, or one or more steps areprovided in a different sequence without departing from the scope of theclaims herein. In certain embodiments, any of these steps may becombined with the others recited herein.

As a thirteenth example, a method for running a chip run using a classiccrystallization method with a designed chip run according to anembodiment of the present invention is provided by the following processflow. Generally, a precondition for this method is that the chip run isalready created and stored in one or more memories of a computing systemas discussed in relation to the tenth example. FIG. 25 is a simplifiedflowchart illustrating operations performed according to an exemplaryembodiment of the present invention. Accordingly, the reference numeralsin the following process flow refer to FIG. 25.

-   -   2510. A first user designs a set of chip runs and save them to        the ready-to-run list in one or more memories of a computing        system (see tenth example for creating a chip run in the        ready-to-run list).    -   2515. Send the new chip run notice to a second user through        email, phone, or the like.    -   2520. The second user starts Experiment Manager Software on an        automated system, which has been described herein. The second        user uses the Experiment Manager or Internet Explorer to find        the details of the new chip runs and print the work order.    -   2525. The second user takes a chip from the stock (generally not        a TOPAZ™ database function).    -   2530. Dispense the reagent and sample into the chip.    -   2535. Use the Experiment Manager to:        -   Associate this chip to a chip run in the ready-to-run list.        -   Enter the physical information for sample and reagent plate    -   2540. Go to a microscope to view the crystallization result and        enter the results to the database through the Score Card, for        example, a TOPAZ™ Score Card. Generally, whenever the user saves        the annotation results, the database will update the “Annotated        by” field of the chip run.    -   2545. Whenever the same user or a different user approves the        annotation, the user will use a Score Card to update the        “Approved by” field of the chip run, and make the chip run        complete.    -   2550. Crystallization Result:        -   For a diffraction chip,        -   2551 a. If there is a good quality crystal:            -   Extract the crystal.            -   Go to the beam. In some embodiments, this step involves                performing crystallography including, but not limited                to, x-ray diffraction crystallography.                -   Structure resolved. The END.                -   Structure not resolved. Repeat the experiment or a                    different reagent set design.        -   2551 b. If there is not a good quality crystal:            -   Repeat the experiment or use a different reagent set                design.        -   2551 c. If there is no crystal:            -   Select a different reagent set and re-run the                experiment.        -   For a screening chip,        -   2552 a. If there is a good quality crystal:            -   From the crystallization condition, create a new reagent                set or select a reagent set for the diffraction run.        -   2552 b. If there is not a good quality crystal:            -   From a set of the crystallization conditions, create a                new reagent set or select a reagent set for an                optimization chip run.        -   2552 c. If there is no crystal:            -   Select a different reagent set and re-run the                experiment.    -   2555. Perform any other steps, as desired.

As shown, above, the above steps can be used to run a chip run using aclassic crystallization method with a designed chip run according to anembodiment of the present invention. Other alternatives can also beprovided where steps are added, one or more steps are removed, or one ormore steps are provided in a different sequence without departing fromthe scope of the claims herein. In certain embodiments, any of thesesteps may be combined with the others recited herein.

As a fourteenth example, a method for running a chip run using a classiccrystallization method without a designed chip run according to anembodiment of the present invention is provided by the following processflow. FIG. 26 is a simplified flowchart illustrating operationsperformed according to an exemplary embodiment of the present invention.Accordingly, the reference numerals in the following process flow referto FIG. 26.

-   -   2610. A second user takes a chip from the stock.    -   2620. Dispense the reagent and sample into the chip.    -   2630. The second user starts Experiment Manager Software on an        automated system, which has been described herein. Start        Experiment Manager Software if desired on an automated system,        which has been described herein. Use the Experiment Manager to        create a new chip run, and then associate the reagent plate and        sample information with the new chip run.        -   Associate this chip to a chip run in the Ready-to-Run list.        -   Enter the physical information for the Sample and the            Reagent Plate.    -   After completion of 3, additional steps as provided by steps        2540 to 2550 in the thirteenth example are followed.    -   2670. Perform any other steps, as desired.

As shown, above, the above steps can be used to run a chip run usingclassic crystallization method without a designed chip run according toan embodiment of the present invention. Other alternatives can also beprovided where steps are added, one or more steps are removed, or one ormore steps are provided in a different sequence without departing fromthe scope of the claims herein. In certain embodiments, any of thesesteps may be combined with the others recited herein.

In certain embodiments, the disclosed invention encompasses a system formanaging workflow related to processing of one or more microfluidicdevices including devices that perform polynucleotide amplifications,immunological reactions, chemical synthesis or degradation reactions, orany chemical or biochemical, organic or inorganic reaction that providesa visibly detectable reaction product.

Microfluidic devices for performing polynucleotide amplifications aredisclosed, for example, in U.S. Patent Application Publication No.2003/0138829, U.S. Patent Application Publication No. 2005/0129581, andU.S. Patent Application Publication No. 2005/0214173, all of which arehereby incorporated by reference for all purposes.

Such devices may comprise one or more elastomeric layers made bymultilayer soft lithography having the plurality of microfluidicchannels and reaction chambers/sites that may be defined and isolated bythe actuation of one or more elastomeric valves. Such devices aredescribed in detail by Unger et al. (2000) Science 288:113-116, in U.S.patent application Ser. No. 09/605,520, filed Jun. 27, 2000, in U.S.patent application Ser. No. 09/724,784, filed Nov. 28, 2000, and in PCTpublication WO 01/01025, all of which are hereby incorporated byreference for all purposes.

In certain embodiments, a microfluidic device for performingpolynucleotide amplifications may include a heat exchange element viawhich heat is exchanged between the reaction chambers and athermocycler. A heat exchange element may comprise a plate or surfacehaving a higher thermal conductivity and/or a lower thermal heatcapacity than the surrounding material of which the microfluidic deviceis fabricated. For instance the body of the microfluidic device may bemade substantially of an elastomeric polymer such as PDMS, and the heatexchange element may be made of silicon or a semiconductor material or ametal such as steel, aluminum or copper. The heat exchange element maybe sized, shaped and placed in contact with the body of the microfluidicdevice such that heat energy may be readily exchanged with the reactionchambers when in use. In certain preferred embodiments the heat exchangeelement is integrally fabricated into the structure of the microfluidicdevice, for example as a plate integrated into the bottom portion of thedevice wherein one side of the plate is in contact with the bottomsurface of the microfluidic device and the other side is exposed so asto be available to be contacted with a complementary thermocycler plate.

In certain embodiments, the disclosed invention encompasses a system formanaging workflow related to processing of one or more microfluidicdevices for which the microfluidic device comprises one or more wellregions, each of the well regions being capable of processing one ormore of the process designs associated with the one or more respectiveprocesses; and wherein the process encompasses a PCR reaction.

In various preferred embodiments, one or more channels within themicrofluidic device is a blind flow channel. Such a blind flow channelmay include a region that functions as a reaction site. Certain suchdevices include a flow channel formed within an elastomeric material,and a plurality of blind flow channels in fluid communication with theflow channel, with a region of each blind flow channel defining areaction site. The device can also include one or more control channelsoverlaying and intersecting one or more of the blind flow channels. Thedevices can optionally further include a plurality of guard channelsformed within the elastomeric material and overlaying the flow channeland/or one or more of the reaction sites. The guard channels aredesigned to have fluid flow therethrough to reduce evaporation from theflow channels and reaction sites of the device. Additionally, thedevices can optionally include one or more reagents deposited withineach of the reaction sites.

In certain devices a single flow channels is in fluid communication withmultiple blind flow channels branching from the flow channel. In someinstances the flow channels are interconnected with one another. Inother devices, however, the plurality of flow channels are isolated fromeach other such that fluid introduced into one flow channel cannot flowto another flow channel, and each flow channel comprises an inlet at oneor both ends into which fluid can be introduced.

In certain embodiments, the architecture of the flow channels and samplechannels is such that the device enables every possible pairwisecombination of a number of reagents and a number of samples. For examplefor samples S1 and S2 and reagents R1 and R2, the architecturefacilitates the separate and uncontaminated mixing of sample and reagentto produce combinations: R1S1, R2S1, R1S2 and R2S2.

Other embodiments encompass a device in which a reagent for conducting areaction is immobilized. The reagent mat be a reagent for conducting anucleic acid amplification reaction such as a primer, polymerase and oneor more nucleotides.

In certain embodiments, the disclosed invention encompasses a system formanaging workflow related to processing of one or more microfluidicdevices including devices that perform immunological reactions.

Microfluidic devices for performing immunological reactions aredisclosed, for example, in U.S. Patent Application Publication No.2004/0072278 and U.S. Patent Application Publication No. 2004/0180377,both of which are hereby incorporated by reference for all purposes.Such devices are usable to conduct immunological experiments and assayssuch as qualitative and quantitative immunological assays includingELISA reactions.

Microfluidic devices for use with the invention include devices with theability to conduct many simultaneous experiments with no cross-talkbetween the antibodies. Cross-talk is eliminated by physically isolatingeach primary and secondary antibody in the panel. Each primary antibodyis delivered through a separate primary delivery channel, into areaction chamber. These reaction chambers are then flowed through with asample containing the target antigen, and then flowed through with asecond antibody which is also separate and delivered via a second inlet.

The isolation and separate delivery of the secondary antibody to thereception chambers is fundamental to eliminating potential cross-talkbetween all antibodies in the panel. Current systems combine thesecondary antibodies into a single cocktail which is used to flood theassay cambers. This cocktail may contain antibodies that will cross-talkwith other antibodies (e.g. secondary antibodies may bindnon-specifically with primary antibodies). This cross-talk problemrequires users to employ the very laborious process of screeningindividual pairs of antibodies against one another to determinecross-talk and therefore identify the “poor quality” antibodies thatproduce the false positives.

The devices of the invention, for example, may be used as an analyticaltool to determine whether a particular target protein or peptide ofinterest is present or absent in a sample. Such devices may also be usedto detect the presence or amount of antibodies in a sample.

The devices may be utilized to test for the presence or quantity ofparticular pathogen, or for the presence or quantity of proteins,peptides or antibodies associated with such pathogens (e.g., viruses,bacteria or fungi). Such applications would provide a quick, efficient,accurate, sensitive, and inexpensive screening method useful in publichealth and counter-bio-terrorism applications. The invention may also beused to detect protein and non-protein agents, poisons and toxins (suchas abrin, ricin and modeccin etc), and nerve agents, e.g., the “G”agents (such as tabun, soman, sarin, and cyclosarin) and the “V” agents,such as VX. Such devices could also be used for identification purposes(e.g., paternity and forensic applications). Such devices could also beutilized to detect or characterize specific proteins or antibodiescorrelated with particular diseases or genetic disorders such asdiabetes, cancer, and the like.

Alternatively, the devices can be used to perform combinatorialsynthetic chemistry or immunology, preparing a large number ofcombinations simultaneously.

According to some embodiments of the present invention, microfluidicdevices provided herein provide reaction chambers characterized by smallpredetermined volumes. Merely by way of example, some devices providechambers with a volume of about 25 μl and less. In a particular,embodiment, chambers with a volume of less than about 1 μl to about 1 nlare provided. The throughput of systems utilizing reaction chamberscharacterized by these small predetermined volumes is higher thanconventional systems. Moreover, as described throughout the presentspecification, numerous combinations of samples and reagents are scannedusing embodiments of the present invention.

Moreover, embodiments of the present invention provide microfluidicdevices characterized by reduced spatial and temporal variation comparedto conventional devices. For example, some microfluidic device providedherein reduce spatial variation as a result on the reduced spatialdimensions of the microfluidic devices in comparison to conventionalmicrotiter plates. Accordingly, temperature variations across thechambers present in the microfluidic device are reduced, enablingincreased accuracy in performing PCR processes. Additionally, thesereduced spatial dimensions result in a decrease in temporal variations,as reactions are initiated and terminated within a smaller time windowthan achieved using conventional techniques such as microtiter plates.One of ordinary skill in the art would recognize many variations,modifications, and alternatives.

Matrix Design

A. General

Devices utilizing the matrix design generally have a plurality ofvertical and horizontal flow channel that intersect to form an array ofjunctions. Because a different sample and reagent (or set of reagents)can be introduced into each of the flow channels, a large number ofsamples can be tested against a relatively large number of reactionconditions in a high throughput format. Thus, for example, if adifferent sample is introduced into each of M different vertical flowchannels and a different reagent (or set of reagents) is introduced intoeach of N horizontal flow channels, then M×N different reactions can beconducted at the same time. Typically, matrix devices include valvesthat allow for switchable isolation of the vertical and horizontal flowchannels. Said differently, the valves are positioned to allow selectiveflow just through the vertical flow channels or just through thehorizontal flow channels. Because devices of this type allow flexibilitywith respect to the selection of the type and number of samples, as wellas the number and type of reagents, these devices are well-suited forconducting analyses in which one wants to screen a large number ofsamples against a relatively large number of reaction conditions. Thematrix devices can optionally incorporate guard channels to help preventevaporation of sample and reactants.

B. Exemplary Designs and Uses

FIG. 27 provides an illustration of one exemplary matrix device. Thisdevice 2700 comprises seven vertical flow channels 2702 and sevenhorizontal flow channels 2704 that intersect to form an array of 49different intersections or reaction sites 2706. This particular devicethus enables seven samples to be reacted with seven different reagentsor sets of reagents. Column valves 2710 that regulate solution flow inthe vertical direction can be controlled by control channels 2718 thatcan all be actuated at a single inlet 2714.

Similarly, row valves 2708 regulate solution flow in the horizontaldirection; these are controlled by control channels 2716 that areactuated by a single control inlet 2712. As shown in FIG. 27, thecontrol channels 2716 that regulate the row valves 2708 vary in widthdepending upon location. When a control channel 2716 crosses a verticalflow channel 2702, the control channel 2716 is sufficiently narrow thatwhen it is actuated it does not deflect into the vertical flow channel2702 to reduce substantially solution flow therethrough. However, thewidth of the control channel 2716 is increased when it overlays one ofthe horizontal flow channels 2704; this makes the membrane of thecontrol channel sufficiently large to block solution flow through thehorizontal flow channel 2704.

In operation, reagents R1-R7 are introduced into their respectivehorizontal flow channels 2704 and samples S1-S7 are injected into theirrespective vertical flow channels 2702. The reagents in each horizontalflow channel 2704 thus mix with the samples in each of the vertical flowchannels 2702 at the intersections 2706, which in this particular deviceare in the shape of a well or chamber. Thus, in the specific case of anucleic acid amplification reaction, for example, the reagents necessaryfor the amplification reaction are introduced into each of thehorizontal flow channels 2704. Different nucleic acid templates areintroduced into the vertical flow channels 2702. In certain analyses,the primer introduced as part of the reagent mixture that is introducedinto each of the horizontal flow channels 104 might differ between flowchannels. This allows each nucleic acid template to be reacted with anumber of different primers.

Blind Channel Designs

A. General

Devices utilizing a blind channel design have certain features. First,the devices include one or more flow channels from which one or moreblind channels branch. As indicated above, the end region of suchchannels can serve as a reaction site. A valve formed by an overlayingflow channel can be actuated to isolate the reaction site at the end ofthe blind channel. The valves provide a mechanism for switchablyisolating the reaction sites.

Second, the flow channel network in communication with the blindchannels is configured such that all or most of the reaction sites canbe filled with a single or a limited number of inlets (e.g., less than 5or less than 10). The ability to fill a blind flow channel is madepossible because the devices are made from elastomeric material. Theelastomeric material is sufficiently porous such that air within theflow channels and blind channels can escape through these pores assolution is introduced into the channels. The lack of porosity ofmaterials utilized in other microfluidic devices precludes use of theblind channel design because air in a blind channel has no way to escapeas solution is injected.

A third characteristic is that one or more reagents are non-covalentlydeposited on a base layer of elastomer during manufacture (see infra forfurther details on the fabrication process) within the reaction sites.The reagent(s) are non-covalently attached because the reagents aredesigned to become dissolved when sample is introduced into the reactionsite. To maximize the number of analyses, a different reactant or set ofreactants is deposited at each of the different reaction sites.

Certain blind channel devices are designed such that the reaction sitesare arranged in the form of an array.

Thus, in those blind channel devices designed for conducting nucleicacid amplification reactions, for example, one or more of the reagentsrequired for conducting the extension reaction are deposited at each ofthe reaction sites during manufacture of the device. Such reagentsinclude, for example, all or some of the following: primers, polymerase,nucleotides, cofactors, metal ions, buffers, intercalating dyes and thelike. To maximize high throughput analysis, different primers selectedto amplify different regions of DNA are deposited at each reaction site.Consequently, when a nucleic acid template is introduced into thereaction sites via inlet, a large number of extension reactions can beperformed at different segments of the template. Thermocycling necessaryfor an amplification reaction can be accomplished by placing the deviceon a thermocycling plate and cycling the device between the variousrequired temperatures.

The reagents can be immobilized in a variety of ways. For example, insome instances one or more of the reagents are non-covalently depositedat the reaction site, whereas in other instances one or more of thereagents is covalently attached to the substrate at the reaction site.If covalently attached, the reagents can be linked to the substrate viaa linker. A variety of linker types can be utilized such asphotochemical/photolabile linkers, themolabile linkers, and linkers thatcan be cleaved enzymatically. Some linkers are bifunctional (i.e., thelinker contains a functional group at each end that is reactive withgroups located on the element to which the linker is to be attached);the functional groups at each end can be the same or different. Examplesof suitable linkers that can be used in some assays include straight orbranched-chain carbon linkers, heterocyclic linkers and peptide linkers.A variety of types of linkers are available from Pierce Chemical Companyin Rockford, Ill. and are described in EPA 188,256; U.S. Pat. Nos.4,671,958; 4,659,839; 4,414,148; 4,669,784; 4,680,338, 4,569,789 and4,589,071, and by Eggenweiler, H. M, Pharmaceutical Agent DiscoveryToday 1998, 3, 552. NVOC (6 nitroveratryloxycarbonyl) linkers and otherNVOC-related linkers are examples of suitable photochemical linkers(see, e.g., WO 90/15070 and WO 92/10092). Peptides that have proteasecleavage sites are discussed, for example, in U.S. Pat. No. 5,382,513.

B. Exemplary Designs and Uses

FIG. 28 is a simplified plan view of one exemplary device utilizing theblind channel design. The device 2800 includes a flow channel 2804 and aset of branch flow channels 2806 branching therefrom that are formed inan elastomeric substrate 2802. Each branch flow channel 2806 terminatesin a reaction site 2808, thereby forming an array of reaction sites.Overlaying the branch flow channels 2806 is a control channel 2810 thatis separated from the branch flow channels 2806 by membranes 2812.Actuation of control channel 2810 causes membranes 2812 to deflect intothe branch flow channels 2806 (i.e., to function as a valve), thusenabling each of the reaction sites 2808 to be isolated from the otherreaction sites.

Operation of such a device involves injecting a test sample into flowchannel 2804 with solution subsequently flowing into each of branchchannels 2806. Once the sample has filled each branch channel 2806,control channel 2810 is actuated to cause activation of valves/membranes2812 to deflect into branch channels 2806, thereby sealing off each ofreaction sites 2808. As the sample flows into and remains in reactionsites 2808, it dissolves reagents previously spotted at each of thereaction sites 2808. Once dissolved, the reagents can react with thesample. Valves 2812 prevent the dissolved reagents at each reaction site2808 from intermixing by diffusion. Reaction between sample and reagentsare then detected, typically within reaction site 2808. Reactions canoptionally be heated as described in the temperature control sectioninfra.

Example 1 Signal Strength Evaluations

I. Introduction

The purpose of this set of experiments was to demonstrate thatsuccessful PCR reactions can be conducted with a microfluidic device ofthe design set forth herein with signal strength greater than 50% of theMacro TaqMan reaction.

II. Microfluidic Device

A three layer microfluidic device, fabricated using the MSL process, wasdesigned and fabricated for conducting the experiments described in thefollowing example; FIG. 29A shows a cross-sectional view of the device.As shown, the device 2900 includes a layer 2922 into which is formed theflow channels. This fluid layer 2922 is sandwiched between an overlayinglayer 2920 that includes the control and guard layers and an underlyingsealing layer 2924. The sealing layer 2924 forms one side of the flowchannels. The resulting three-layer structure is affixed to a substrate2926 (in this example, a slide or coverslip), which provides structuralstiffness, increases thermal conductivity, and helps to preventevaporation from the bottom of microfluidic device 2900.

FIG. 29B shows a schematic view of the design of the flow channels inflow layer 2922 and of the control channels and guard channel incontrol/guard layer 2920. Device 2900 consists of ten independent flowchannels 2902, each with its own inlet 2908, and branching blindchannels 2904, each blind channel 2904 having a 1 nl reaction site 2906.Device 2900 contains a network of control lines 2912, which isolate thereaction sites 2906 when sufficient pressure is applied. A series ofguard channels 2916 are also included to prevent liquid from evaporatingout of the reaction sites 2906; fluid is introduced via inlet 2918.

II. Experimental Setup

A PCR reaction using 13-actin primers and TaqMan probe to amplify exon 3of the β-actin gene from human male genomic DNA (Promega, Madison Wis.)was conducted in device 2900. The TaqMan reaction consists of thefollowing components: 1× TaqMan Buffer A (50 mM KCl, 10 mM Tris-HCl,0.01M EDTA, 60 nM Passive Reference 1 (PR1), pH 8.3); 3.5-4.0 mM MgCl;200 nM dATP, dCTP, dGTP, 400 nM dUTP; 300 nM β-actin forward primer andreverse primer; 200 nM FAM-labeled β-actin probe; 0.01 U/μl AmpEraseUNG(Applied Biosystems, Foster City, Calif.); 0.1-0.2 U/μl DyNAzyme(Finnzyme, Espoo, Finland); 0.5% Triton-x-100 (Sigma, St. Louis, Mo.);0.8 μg/μl Gelatin (Calbiochem, San Diego, Calif.); 5.0% Glycerol (Sigma,St. Louis, Mo.); deionized H₂O and male genomic DNA. The components ofthe reaction were added to produce a total reaction volume of 25 μl.Negative controls (Control) composed of all the TaqMan reactioncomponents, except target DNA were included in each set of PCRreactions.

Once the TaqMan reaction samples and Control were prepared, they wereinjected into microfluidic device 2900 by using a gel loading pipet tipattached to a 1 ml syringe. The pipet tip was filled with the reactionsamples and then inserted into the fluid via 2908. The flow channels2902 were filled by manually applying backpressure to the syringe untilall the entire blind channels 2904 and reaction sites 2906 were filled.Control lines 2912 were filled with deionized water and pressurized to15-20 psi after all of the samples were loaded into the flow lines 2902,2904. The pressurized control lines 2912 were actuated to close thevalves and isolate the samples in the 1 nl wells 2906. The guardchannels 2916 were then filled with deionized water and pressurized to5-7 psi. Mineral oil (15 μl) (Sigma) was placed on the flatplate of athermocycler and then the microfluidic device/coverglass 2900 was placedon the thermocycler. Micro fluidic device 2900 was then thermocycledusing an initial ramp and either a three-step or two-step thermocyclingprofile:

-   -   1. Initial ramp to 95° C. and maintain for 1 minute (1.0° C./s        to 75° C., 0.1° C./sec to 95° C.).    -   2. Three step thermocycling for 40 cycles (92° C. for 30 sec.,        54° C. for 30 sec., and 72° C. for 1 min) or;    -   3. Two step thermocycling for 40 cycles (92° C. for 30 seconds        and 60° C. for 60 sec.)

MicroAmp tubes (Applied Biosystems, Foster City, Calif.) with theremaining reaction mixture, designated Macro TaqMan reactions todistinguish them from reactions performed in the microfluidic device,were placed in the GeneAmp PCR System 9700 (Applied Biosystems, FosterCity, Calif.) and thermocycled in the 9600 mode. The Macro TaqManreactions served as macroscopic controls for the reactions performed inthe micro fluidic device. The thermocycling protocol was set to matchthat of the microfluidic device, except that the initial ramp rate wasnot controlled for the Macro TaqMan reactions.

Once thermocycling was completed, the control and guard lines weredepressurized and the chip was transferred onto a glass slide (VWR, WestChester, Pa.). The chip was then placed into an Array WoRx Scanner(Applied Precision, Issaquah, Wash.) with a modified carrier. Thefluorescence intensity was measured for three differentexcitation/emission wavelengths: 475/510 nm (FAM), 510/560 nm (VIC), and580/640 nm (Passive Reference 1 (PR1)). The Array Works Software wasused to image the fluorescence in the micro fluidic device and tomeasure the signal and background intensities of each 1 nl well. Theresults were then analyzed using a Microsoft Excel file to calculate theFAM/PR1 ratio for β-actin TaqMan reactions. For conventional MacroTaqMan, positive samples for target DNA were determined usingcalculations described in the protocol provided by the manufacturer(TaqMan PCR Reagent Kit Protocol). The signal strength was calculated bydividing the FAM/PR1 ratio of the samples by the FAM/PR1 ratio of thecontrols. A successful reaction was defined as a sample ratio above the99% confidence threshold level.

III. Results

Initially, AmpliTaq Gold (Applied Biosystems, Foster City, Calif.) wasused in TaqMan reactions and FAM/PR1/Control ratios of 1.5-2.0 wereproduced, compared to Macro TaqMan reaction ratios of 5.0-14.0. Althoughresults were positive, increased signal strength was desired. Therefore,the AmpliTaq Gold polymerase was substituted with DyNAzyme polymerasedue to its increased thermostability, proofreading, and resistance toimpurities. The standard Macro TaqMan DyNAzyme concentration of 0.025U/μl was used in the microfluidic experiments. This polymerase change toDyNAzyme produced FAM/ROX/Control ratios of 3.5-5.8. The signal strengthwas improved, but it was difficult to achieve consistent results.Because it is know that some proteins stick to PDMS, the concentrationof the polymerase was increased and surface modifying additives wereincluded. Two increased concentrations of DyNAzyme were tested, 8×(0.2U/μl) and 4×(0.1 U/μl) the standard concentration for Macro TaqMan, with100 pg or 10 pg of genomic DNA per nl in the micro fluidic device.Gelatin, Glycerol, and 0.5% Triton-x-100 were added to prevent thepolymerase from attaching to the PDMS.

The microfluidic TaqMan reaction ratios range from 4.9-8.3, while theMacro TaqMan reactions range from 7.7-9.7. Therefore, the signalstrength of the TaqMan reactions in chip is up to 87% of the MacroTaqMan reactions. There was no significant difference between 4× or 8×DyNAzyme. The results demonstrate that PCR reactions can be done withgreater than 50% signal strength, when compared to the Macro TaqManreactions, in the microfluidic devices. The results have been consistentthrough at least four attempts.

Example 2 Verification of PCR by Gel Electrophoresis

I. Introduction

As an alternative method to prove amplification of DNA was occurring inthe microfluidic device, an experiment to detect PCR product by gelelectrophoresis was performed. PCR reactions compositions were asdescribed in Example 1, except the TaqMan probe was omitted and theβ-actin forward primer was conjugated to FAM.

II. Procedure

A. Microfluidic Device

A three layer microfluidic device, fabricated using the MSL process, wasdesigned and fabricated for conducting the experiments described in thisexample; FIG. 30 shows a schematic view of the design. The device 3000generally consists of a sample region 3002 and a control region 3004.Sample region 3002 contains three hundred and forty-one 1 nl reactionsites 3008 represented by the rectangles arrayed along flow channel3006, which includes inlet via 3010 and outlet via 3012. Control region3004 contains three control flow channels 3014 each containing ten 1 nlreaction sites 3018, also represented by the rectangles and an inlet via3016. A network of control lines 3022 isolate each reaction site 3008,3018 when sufficient pressure is applied to inlet via 3024. A series ofguard channels 3020 are included to prevent liquid from evaporating outof the reaction sites 3008, 3018. The device is a three-layer device asdescribed in Example 1 (see FIG. 29A). The entire chip is placed onto acoverslip.

B. Experimental Setup

Microfluidic device 3000 was loaded and thermocycled using the 3temperature profile described in Example 1. The remaining reactionsample was thermocycled in the GeneAmp 9700 with the same thermocyclingprofile as for microfluidic device 3000. The reaction products wererecovered after thermocycling was completed. To recover the amplifiedDNA, 3 μl of water was injected into sample input via 3006 and 3-4 μl ofproduct were removed from outlet via 3012. The reaction products fromdevice 3000 and the Macro reaction were treated with 2 μl of ExoSAP-IT(USB, Cleveland, Ohio), which is composed of DNA Exonuclease I andShrimp Alkaline Phosphatase, to remove excess nucleotides and primers.The Macro product was diluted from 1:10 to 1:106. The product fromdevice 3000 was dehydrated and resuspended in 4 μl of formamide.

III. Results

Both products, along with negative controls were analyzed, on apolyacrylamide gel. FIG. 30 shows the gel electrophoresis results. Theappropriate size DNA band of 294 base pairs in length was observed.

The products from the Macro reactions are shown on the left hand side ofthe gel and correspond to about 294 base pairs, the expected size of theβ-actin PCR product. The negative controls lack the PCR product.Similarly, the product derived from the device gave the expected β-actinPCR product. Therefore, target DNA was amplified in the micro fluidicdevice.

Example 3 Massive Partitioning

The polymerase chain reaction (PCR) has become an essential tool inmolecular biology. Its combination of sensitivity (amplification ofsingle molecules of DNA), specificity (distinguishing single basemismatches) and dynamic range (10⁵ with realtime instrumentation) makeit one of the most powerful analytical tools in existence. Wedemonstrate here that PCR performance improves as the reaction volume isreduced: we have performed 21,000 simultaneous PCR reactions in a singlemicrofluidic chip, in a volume of 90 pL per reaction and with singletemplate molecule sensitivity.

FIGS. 31A-31D depict a single bank and dual bank partitioningmicrofluidic device where multilayer soft lithography (MSL) (1), wasused to create elastomeric microfluidic chips which use active valves tomassively partition each of several liquid samples into a multitude ofisolated reaction volumes. After injection of the samples into inlet3103 which is in communication with branched partitioning channel system3105 of microfluidic device 3101 (FIG. 31B), 2400 90 pL volumes 3109 ofeach sample are isolated by closing valves 3107 spaced along (FIG. 31D)simple microfluidic channels. The chip device is then thermocycled on aflat plate thermocycler and imaged in a commercially availablefluorescence reader.

FIG. 32 is a schematic drawing of one “immunochip” embodiment of thedevice having 24 sample inputs and 24 primary antibody inputs, and 24secondary antibody inputs. Additional disclosure related to immunochipembodiments of the present invention are provided in U.S. ProvisionalPatent Application No. 60/716,823, entitled “Microfluidic Devices forPerforming Immunological Assays,” filed on Sep. 13, 2005, andincorporated herein by reference for all purposes.

FIG. 33 is a schematic drawing of another embodiment of the devicehaving 96 sample inputs and 24 primary antibody inputs, and 24 secondaryantibody inputs.

FIG. 34 is a schematic drawing showing a magnified section of anembodiment the device. The primary inlet is the inlet through which theprimary antibody is introduced. The secondary is the inlet through whichthe secondary antibody is introduced. The wash inlet is the inletthrough which washing solution is introduced. The two waste outlets arethe outflows through which the excess reactant fluid (of any kind) isexpelled. In use, the blocking solution is flowed through the lumens ofthe chip via the common inlet. The primary antibody solution is thenintroduced through the primary inlet and flows through the lumens of thechip and through the reaction chambers. A wash solution is then flowedthrough all the lumens and chambers to remove unbound primary antibody.The secondary antibody solution is introduced through the secondaryinlet and flows through the lumens of the chip and through the reactionchambers. Another wash solution is then flowed through all the lumensand chambers to remove unbound secondary antibody. The enzyme solutionis then introduced, followed by another washing step, followed by thesubstrate solution, followed by a final wash step. A signal is thusproduced and measured.

According to embodiments of the present invention, in addition toprotein crystallization experiments, there are multiple examples ofother fluorogenic reactions, chemiluminescent reactions, colorimetricreactions, and radiometric reactions that are run utilizing the DatabaseApplication Suite. Some of these experiments involve multiple softwarecomponents, while others will only involve one component. One ofordinary skill in the art would recognize many variations,modifications, and alternatives. Additionally, some of these examples ofusing methods and systems according to embodiments of the presentinvention do not constitute performance of entire experiments, butmerely portions or sub-elements thereof.

FIG. 35 is a simplified flowchart illustrating a method of performinggene expression/genotyping experiments according to an embodiment of thepresent invention. As shown in FIG. 35, several different experimentsmay be performed in a sequential and/or recursive manner according toembodiments of the present invention.

FIGS. 36-40 are simplified flowcharts illustrating operations performedaccording to exemplary embodiments of the present invention.

As a first example, a method for using a Dynamic Array, for example a48×48 element array, for gene expression analysis according to anembodiment of the present invention is provided by the following processflow. FIG. 36 is a simplified flowchart illustrating operationsperformed according to an exemplary embodiment of the present invention.Accordingly, the reference numerals in the following process flow referto FIG. 36.

-   -   3610. A first user acquires biological samples, develops,        extracts RNA, and processes RNA into cDNA. In some embodiments,        this is an optional step. Additionally, the first user develops        screening reagents (e.g., TaqMan assays).    -   3612. The first user mixes up to 48 samples with real-time PCR        Master Mix and loads sample-master mix mixtures into the sample        wells of the 48×48 Dynamic Array. In some embodiments, this step        is performed manually by hand-pipetting, whereas in other        embodiments, this step is performed automatically with automated        liquid handling workstations. One of ordinary skill in the art        would recognize many variations, modifications, and        alternatives.    -   3614. The first user loads screening reagents into the reagent        wells of the 48×48 Dynamic Array. In some embodiments, this step        is performed manually by hand-pipetting, whereas in other        embodiments, this step is performed automatically with automated        liquid handling workstations.    -   3616. The 48×48 Dynamic Array is placed into a chip loading        instrument (e.g., a NanoFlex IFC Controller) by the first user.        In some embodiments, a FIDX™ is utilized for this step.    -   3618. The NanoFlex IFC Controller loads the sample-master mix        mixtures and screening reagents from the wells of the 48×48        Dynamic Array into the appropriate reactions in the integrated        fluidic circuit (IFC) of the 48×48 Dynamic Array. According to        an embodiment of the present invention, a chip-specific computer        script controls this loading operation.    -   3620. The 48×48 Dynamic Array is moved from the NanoFlex IFC        Controller to the BioMark System. In a particular embodiment,        this step involves placing the 48×48 Dynamic Array in a chip        tray such as a microwell plate tray).    -   3622. The orientation of the 48×48 Dynamic Array is verified.    -   3624. The chip tray (e.g., the microwell plate tray), controlled        by a motor, draws the 48×48 Dynamic Array into the BioMark        System.    -   3626. A barcode reader reads the barcode on the 48×48 Dynamic        Array. According to embodiments of the present invention, each        barcode contains a unique number to identify the 48×48 Dynamic        Array as well as the chip type (e.g. 48×48 Dynamic Array,        digital array, and the like).    -   3628. The microwell plate tray, with the 48×48 Dynamic Array        inside, stops at a position above a thermal cycler.    -   3630. A motor controls vertical movement of the thermal cycler.        The motor moves the thermal cycler up until a vacuum seal        between the thermal block and an IHS (integrated heat spreader)        of the 48×48 Dynamic Array is detected.    -   3632. Autofocusing (typically performed by moving the thermal        cycler up or down) normal to the chip tray, is used to focus the        optical system.    -   3634. Fluorescence intensity of test images are used to set an        intensity of one or more xenon lamps for each wavelength of        interest.    -   3636. Thermal cycling commences. According to embodiments of the        present invention, protocols are pre-programmed or entered prior        to the run.    -   3638. Images are taken with a CCD camera during the thermal        cycling protocol. The one or more xenon lamps, excitation filter        wheel, emission filter wheel, thermal cycler, CCD camera, and        computer are synchronized in an embodiment in order to properly        collect images on all wavelengths of interest.    -   3640. After the run is complete, the microwell plate tray moves        the 48×48 Dynamic Array out of the BioMark System.    -   3642. In a specific embodiment, steps 3616-3640 are repeated in        a batch mode. In exemplary embodiments, batch mode operation is        performed provided that multiple 48×48 Dynamic Arrays were set        up in steps 3612 and 3614 and that automation is in place for        the steps that are external to the BioMark System.    -   3644. Software analysis commences as described in more detail        throughout the present specification.    -   3646. Perform any other steps, as desired.

As shown, above, the above steps can be used to use a Dynamic Array forgene expression analysis according to an embodiment of the presentinvention. Other alternatives can also be provided where steps areadded, one or more steps are removed, or one or more steps are providedin a different sequence without departing from the scope of the claimsherein. In certain embodiments, any of these steps may be combined withthe others recited herein.

As a second example, a Matrix Instrument is used to set up and run aDynamic Array Gene Expression experiment. Various numbers of reactionchambers (e.g., 48×48 or 96×96) are provided herein. Preferably, anumber of initial conditions are provided prior to running the geneexpression experiment. These initial conditions may include: theInstrument System is installed; the Instrument System is calibrated; theInstrument System is powered up and warmed up; the FIDX™ System isinstalled; and the user has prepared cDNA and assays; the user hasdesigned the experiment. FIG. 37 is a simplified flowchart illustratingoperations performed according to an exemplary embodiment of the presentinvention. Accordingly, the reference numerals in the following processflow refer to FIG. 37.

-   -   3710. The User moves carrier into FIDX.    -   3712. User pressurizes chip interface and containment fluid        using FIDX.    -   3714. User removes carrier from FIDX.    -   3716. User moves carrier to reagent loading area.    -   3718. User loads carrier I/O ports with cDNA samples and TaqMAN        assays. This step may be performed either manually or via a        robotic system.    -   3720. User manually types sample/reagent information (map), and        thermal cycling protocol into software (e.g. experiment        document).    -   3722. Instrument verifies that protocol information is valid.    -   3724. User moves carrier back to FIDX.    -   3726. User initiates FIDX script to load samples and reagents to        chip's sample wells.    -   3728. User removes carrier from FIDX and transports to Dynamic        Array Instrument. Instrument records barcode information.    -   3730. User enters thermal cycler protocol into        software/experiment document.    -   3732. User initiates a run (i.e., starts the method). In some        embodiments, this step will include the various steps discussed        below in relation to FIG. 38.    -   3734. Instrument reports to user the time remaining in the        method.    -   3736. Run completes and instrument informs the user.    -   3738. Carrier is removed from instrument.    -   3740. User analyzes the run data.    -   3742. Data analysis and associated output is saved.    -   3746. Perform other steps as necessary.

As shown, above, the above steps can be used to use a Dynamic Array forgene expression analysis according to an embodiment of the presentinvention. Other alternatives can also be provided where steps areadded, one or more steps are removed, or one or more steps are providedin a different sequence without departing from the scope of the claimsherein. In certain embodiments, any of these steps may be combined withthe others recited herein.

Various alternatives and variations are provided according toembodiments of the present invention. Merely by way of example, in analternative embodiment, at one or more of the steps, a robotic system isutilized to perform the various operations. In another embodiment, atstep 3718, barcode information is read from carrier by a robotic systemand transferred to software. Moreover, at step 3720, the sample andthermal cycler protocol information may be automatically transferred tosoftware/experiment document via the robotic system or barcode. If, atstep 3722, invalid thermal cycler protocol is entered, the instrumentreports the error to the user, requests a valid protocol, and thenreturns to step 3720. In another alternative embodiment, at step 3738,operating in a batch mode, the carrier is removed and the next carrieris loaded. Then the run returns to step 3710. As yet another alternativefor batch mode operation, at step 3738, the system will determine, basedon the time remaining in the currently running method, when to initiateprocessing of the next experiment plate. Merely by way of example, thisdetermination may be made with 60 minutes remaining in current run.Generally, after completion of a run, the carrier is removed and theinstrument is maintained in an idle state.

According to embodiments of the present invention, considerationsrelated to the user interface and data analysis options are provided asdescribed more fully throughout the present specification. Theseconsiderations include the data output format, data persistence,loading, mapping, and displaying of sample information, among others.One of ordinary skill in the art would recognize many variations,modifications, and alternatives.

As a third example, a generic instrument run with a carrier is performedaccording to embodiments of the present invention. Preferably, a numberof initial conditions are provided prior to running the genericinstrument experiment. These initial conditions may include: theInstrument System is installed; the Instrument System is calibrated; theInstrument System is powered up and warmed up; the user has achip/carrier loaded with a sample and ready for the instrument run, thethermal cycler is at an idle state, and the command to load the sampleand run the instrument has been initiated. FIG. 38 is a simplifiedflowchart illustrating operations performed according to an exemplaryembodiment of the present invention. Accordingly, the reference numeralsin the following process flow refer to FIG. 38.

-   -   3810. Sample information is transferred to the instrument        software (Manually or by automation). This preferably includes        loading map, assay type, chip carrier type, run protocol.    -   3812. Instrument verifies sample info is valid (e.g. Method        type, Thermal Cycler protocol).    -   3814. Instrument verifies that there is sufficient disk space        and write permissions to execute requested.    -   3816. Chip carrier is placed on to the instrument's transfer        arm.    -   3818. The load sequence is initiated.    -   3820. Instrument turns on excitation source and initiates warm        up counter.    -   3822. The instrument closes the gripper.    -   3824. Instrument assesses if carrier is correctly positioned.    -   3826. Instrument moves transfer axis arm to in position (in        instrument).    -   3828. Instrument assesses plate type, checks validity against        protocol information and verifies loading arm move completed.    -   3830. Instrument transfers chip/carrier to nominal, focused run        position. In an embodiment, this step includes a number of        sub-steps:        -   3830 a. z-stage moves Cycler up to “hand-off” z position        -   3830 b. Instrument registers carrier against gripper datum            location        -   3830 c. Instrument releases gripper        -   3830 d. Instrument verifies that vacuum vent valve is closed        -   3830 e. Instrument applies vacuum to chuck/carrier interface        -   3830 f. Instrument verifies that vacuum is established        -   3830 g. Instrument moves Cycler to nominal focus z position            (specific to Chip type)        -   3830 h. Instruments sets Thermal Cycler to “set-up”            temperature (e.g. 20° C.)    -   3832. Instrument performs auto-focus routine. In an embodiment,        this step includes a number of sub-steps:        -   3832 a. Instrument moves Excitation and Emission filter            wheels to correct filter position for focus (Uses passive            reference dye channel)        -   3832 b. Instrument determines best exposure time for focus            (“Good” signal/no saturation)        -   3832 c. Instrument moves z stage up and down±n steps and            takes images every “m” steps        -   3832 d. Instrument evaluates images to determine best focus            z position for passive reference channel        -   3832 e. Instrument verifies focus position validity    -   3834. Instrument determines optimal exposure times based on        method and chip/carrier type.    -   3836. Instrument sets optimal focus z-position off-sets        (predetermined for “set-up” temperature) for remaining filter        pairs.    -   3838. Instrument verifies validity of focus off-sets in all        filters required for method.    -   3840. Instrument initiates “Run Method.” For Gene Expression,        the process described in relation to FIG. 39 is utilized in some        embodiments including Real-Time PCR Thermal Cycler Run & Data        Collection. For Genotyping, the process described in relation to        FIG. 40 is utilized in some embodiments, including Genotyping        Run & Data Collection.    -   3842. Method completes.    -   3844. Instrument sets Thermal Cycler to idle.    -   3846. Post run reference images are collected.    -   3848. Software performs validity checks on data.    -   3850. Instrument Ejects Chip/Carrier. In an embodiment, this        step includes a number of sub-steps:        -   3850 a. Instrument turns off vacuum pump        -   3850 b. Instrument opens vacuum vent valve        -   3850 c. Instrument waits/confirms vacuum is vented        -   3850 d. Instrument lowers cycler to “down” z-position        -   3850 e. Instrument closes vacuum vent valve        -   3850 f. Instrument engages gripper on chip/carrier        -   3850 g. Instrument verifies chip/carrier is positioned            correctly        -   3850 h. Instrument moves transfer axis arm to out position        -   3850 i. Instrument verifies move is complete        -   3850 j. Instrument releases gripper    -   3852. Instrument indicates method is complete.    -   3854. The chip/carrier is removed from the transfer arm.    -   3856. Perform other steps as necessary.

As shown, above, the above steps can be used to use a Dynamic Array forgene expression analysis according to an embodiment of the presentinvention. Other alternatives can also be provided where steps areadded, one or more steps are removed, or one or more steps are providedin a different sequence without departing from the scope of the claimsherein. In certain embodiments, any of these steps may be combined withthe others recited herein.

Various alternatives and variations are provided according toembodiments of the present invention. Merely by way of example, in analternative embodiment, at one or more of the steps, automationaccessories are utilized to perform the various operations. Moreover, invarious embodiments, at step 3830, focus optimization is performedmanually, at step 3812, if the assay method validity fails, theinstrument informs user of specific error and T.U.C.E., at step 3814, ifthere is not enough disk space/no read write permission, the instrumentinforms user of specific error and T.U.C.E., at step 3824, if theChip/carrier is positioned incorrectly, the instrument informs user ofspecific error and T.U.C.E., and at step 3828, if the Chip/carrier typedoes not match Assay method type, the instrument informs user ofspecific error and T.U.C.E. Furthermore in some embodiments, at step3828, if the transfer arm move failed, the instrument retries themovement in step 3826 once and then proceeds, otherwise the instrumentinforms user of specific error and T.U.C.E., at step 3830(f), if vacuumis not established, the instrument informs user of specific error andT.U.C.E., at step 2832(e), if the instrument focus position is notvalid, the instrument retries once (goes to 2830(f)), else informs userof specific error and T.U.C.E., and at step 3838, if the focusz-position off-sets are not valid, the instrument informs user ofspecific error and T.U.C.E. Additionally, in other embodiments, at step3848, if the instrument determines errors with data, the user isinformed of specifics and operation proceeds to step 3850, at step3850(c), if vacuum is not released, the instrument proceeds tostep—Instrument goes to 3850(a); at step 3850(g), if the chip/carrier isnot positioned correctly, the instrument retries once (goes to 3850(d)),else informs user of specific error and T.U.C.E., at step 3850(i), ifthe transfer arm did not complete eject move, the instrument retriesonce (goes to 3850(h)), else informs user of specific error and T.U.C.E.

In an alternative embodiment, dosimeter operation is provided. In thisalternative embodiment, pre-set or calibrated information is includedfor each instrument and for each excitation filter position. Thisinformation includes dose monitor setup information (e.g. gain resistor,selectable), which is stored in a computer-readable file, such as an.ini file, during instrument calibration.

Generally, several steps are used to obtain and store dose reading forall cases. These steps are implemented by modifying image acquisitionsteps. (e.g., steps 3912 and 3916 as described in relation to FIG. 39below; and step 4014 as described in relation to FIG. 40 below). Thesteps to obtain and store dose reading for all cases include:

-   -   a. Prepare dose monitor: at same step as camera reset/arm        (discharge capacitor, select range resistor, and the like).    -   b. Start integration/sampling before excitation shutter        commanded to open.    -   c. End integration/sampling after excitation shutter commanded        to close.    -   d. Read out from dose monitor (NI analog read).    -   e. Discharge the dose monitor (in case discharge time helps).    -   f. Log the dose monitor readout with other parameters in        CaptureLog.txt.

According to embodiments of the present invention, the dose monitor isutilized during exposure setting (once per image type: see step 3834 inFIG. 38). This operation may include evaluating the dose reading forunder/over-saturation and reselecting the dose range if needed.Presumably, this will entail re-acquiring an image (or prompting for anew “Test Capture”) if the range needs changing. Also, as a possiblealternative to providing a baseline image as described below, the dosemonitor value may be stored as a basis for normalizing this image typefor this run. The dose monitor may also be utilized to collect baselineimages at start and end of protocol (see step 3910 discussed in relationto FIG. 39 below and step 3846 in relation to FIG. 38. Moreover, thedose monitor may be utilized during cycling, in particular at eachacquisition. Generally, during analysis, the dose reading is retrievedfor a baseline image (e.g. 20° C. pre-run image) and for each well,after ripping and background subtraction, the signal value is scaled bythe ratio of the baseline dose to the current image's dose.

After the above process is complete, the transfer arm is in the “out”position, the chip/carrier is removed, and the instrument is idle. Ingeneral, the Universal Thermal Cycler protocol for Gene Expressionshould be considered and Real Time Display is provided as described morefully throughout the present specification. In relation to step 3814,for a batch process, the check should include disk space and writepermissions for the entire batch, not just the first plate. Moreover, atstep 3834, exposure times are dependent on assay/chemistry type, benchtime, chip type, thermal protocol, chip quality, and the like. In anembodiment, at step 3838, the “Run method” will generate additionalexperiments specific to gene Expression, DID, Immunoassay, and the like.One of ordinary skill in the art would recognize many variations,modifications, and alternatives.

According to embodiments of the present invention, the sampleinformation required by the instrument at the start of a run to meetnecessary traceability and validity requirements is determined.Moreover, 24 hour walk away automation function is provided herein andanalysis is performed and integrated with the instrument controlpackages as described more fully throughout the present specification.Additionally, an exposure time selection methodology is provided byembodiments of the present invention, including a method to selectexposure times for specific protocols/chips.

As a fourth example, a real-time PCR thermal cycler run and datacollection is performed according to embodiments of the presentinvention. Preferably, a number of initial conditions are provided priorto performing a real-time PCR thermal cycler run and data collection.These initial conditions may include the prior initiation of either orboth of the processes described in relation to FIG. 36, 37, or 38.Additionally, initial conditions may include: Chip is loaded ininstrument; Camera/Z-Stage are focused (off sets are established);Exposure times are configured; Excitation source is on and stable;Thermal Cycler is at “set up” temp; Assay/chip information and ThermalCycler protocol information has been “transferred” to the computer (nnumber of cycles); and Command to run thermal cycler protocol has beeninitiated. Typically, a real-time PCR thermal cycler run and datacollection is performed after the Gene Expression experiment is loadedin the instrument and ready to run. FIG. 39 is a simplified flowchartillustrating operations performed according to an exemplary embodimentof the present invention. Accordingly, the reference numerals in thefollowing process flow refer to FIG. 39.

-   -   3910. Instrument collects base line images (see steps 3912,        3916(g), 3916(i), 3916(j), and 3916(k) below)    -   3912. Instrument configures hardware for next data acquisition        point        -   3912 a. Instrument sets excitation filter position        -   3912 b. Instrument sets emission filter position        -   3912 c. Instrument sets Z stage height (offset)    -   3914. Instrument runs initial thermal cycler conditions (e.g.        hot start)        -   3914 a. Instrument sets thermal cycler set point 1 and ramp            power (e.g. 50° C.)        -   3914 b. Instrument queries thermal cycler for “chuck”            temperature        -   3914 c. Once thermal cycler has reached set point            temperature, instrument counts down hold time for set point            1        -   3914 d. Once hold time count has elapsed, Instrument sets            thermal cycler set point 2 and ramp power (e.g. 95° C.)        -   3914 e. Once thermal cycler has reached set point            temperature, instrument counts down hold time for set point            2        -   3914 f. Once hold time count has elapsed instrument runs PCR            cycling method (Or runs further initial cycling condition            set points)    -   3916. Instrument runs n PCR cycles        -   3916 a. Instrument sets thermal cycler initial cycling set            point (e.g. annealing) and ramp power (e.g. 60° C.)        -   3916 b. Instrument queries thermal cycler for “chuck”            temperature        -   3916 c. Once thermal cycler has reached set point            temperature:            -   i. Instrument calculates required data acquisition time                (i.e. total of all filter moves, exposure times and                associated overhead)            -   ii. Instrument subtracts data acquisition time from                total hold time (the result is the pre data acquisition                hold time)        -   3916 d. Instrument counts down remaining pre data            acquisition hold time        -   3916 e. Instrument verifies filter wheel positions and Z            height (Runs step 3912)        -   3916 f. Once pre data acquisition time has elapsed            Instrument requests CCD camera to “reset” and “arm”        -   3916 g. Instrument requests CCD to initiate integration and            sends shutter task to NI card            -   i. NI waits 100 ms            -   ii. NI requests emission shutter to open            -   iii. NI waits 100 ms            -   iv. NI requests excitation shutter to open            -   v. NI waits predetermined exposure time (t ms)            -   vi. NI requests excitation shutter to close            -   vii. waits 100 ms            -   viii. NI requests emission shutter to close        -   3916 h. Instrument sets thermal cycler to second cycling set            point (e.g. denature/95° C.) and ramp power (e.g. 10)        -   3916 i. Instrument requests CCD to transfer (reads out) data        -   3916 j. Instrument requests hardware status (e.g. filter            positions, shutter times, array temp, CCD temp, Z position,            # of data points, heat sink temp . . . )        -   3916 k. Instrument runs step 3912        -   3916 l. Data is saved to TIFF and log (csv) files (Data            Analysis and Real-Time display are additional use cases, at            this point)        -   3916 m. Instrument queries thermal cycler for “chuck”            temperature        -   3916 n. Once thermal cycler has reached set point            temperature, instrument counts down hold time for denature            set point        -   3916 o. Once hold time count has elapsed go to step 3916(a)            (Instrument repeats for n cycles)    -   3918. Once n cycles completes the method ends (See discussion        related to process described with reference to FIG. 38)    -   3920. Perform other steps as necessary.

As shown, above, the above steps can be used to perform real-time PCRthermal cycling and data collection according to an embodiment of thepresent invention. Other alternatives can also be provided where stepsare added, one or more steps are removed, or one or more steps areprovided in a different sequence without departing from the scope of theclaims herein. In certain embodiments, any of these steps may becombined with the others recited herein.

According to embodiments of the present invention, after the real-timePCR method run is complete, and the data is collected, the data is savedand made ready for analysis of the present or future runs. Additionally,24 hour walk away operation is provided by embodiments. Generally, thethermal cycler method run is based on current system architecture andtwo temperature PCR cycling is utilized. Alternatively, otheralternatives are added as appropriate at step 3916(o). Moreover, sampletemperature off set compensation is included in some embodiments. One ofordinary skill in the art would recognize many variations,modifications, and alternatives.

As a fifth example, a genotyping run and data collection is performedaccording to embodiments of the present invention. Preferably, a numberof initial conditions are provided prior to performing a genotyping runand data collection. These initial conditions may include the priorinitiation of either or both of the processes described in relation toFIG. 36, 37, or 38. Additionally, initial conditions may include: Chipis loaded in instrument; Camera/Z-Stage are focused (off sets areestablished); Exposure times are configured; Excitation source is on andstable; Thermal Cycler is at “set up” temp; Assay/chip information andThermal Cycler protocol information has been “transferred” to thecomputer (n number of cycles); and Command to run thermal cyclerprotocol has been initiated. Typically, a genotyping run and datacollection is performed after the Gene Expression experiment is loadedin the instrument and ready to run. FIG. 40 is a simplified flowchartillustrating operations performed according to an exemplary embodimentof the present invention. Accordingly, the reference numerals in thefollowing process flow refer to FIG. 40.

-   -   4010. Instrument runs pre/post read thermal cycler condition        -   4010 a. Instrument sets thermal cycler to data acquisition            set point and ramp power (e.g. 60° C.)        -   4010 b. Instrument queries thermal cycler for “chuck”            temperature        -   4010 c. Once thermal cycler has reached set point            temperature, instrument counts down hold time for data            acquisition stability    -   4012. Instrument configures hardware for next n data acquisition        point        -   4012 a. Instrument sets excitation filter position        -   4012 b. Instrument sets emission filter position        -   4012 c. Instrument sets Z stage height (offset)    -   4014. Instrument initiates data collection sequence        -   4014 a. Instrument requests CCD camera to “reset” and “arm”        -   4014 b. Instrument requests CCD to initiate integration and            sends shutter task to NI card            -   i. NI waits 100 ms            -   ii. NI requests emission shutter to open            -   iii. NI waits 100 ms            -   iv. NI requests excitation shutter to open            -   v. NI waits predetermined exposure time (t ms)            -   vi. NI requests excitation shutter to close            -   vii. NI waits 100 ms            -   viii. NI requests emission shutter to close        -   4014 c. Instrument requests CCD to transfer (reads out) data        -   4014 d. Instrument requests hardware status (e.g., filter            positions, shutter times, array temp, CCD temp, Z position,            # of data points, heat sink temp, and the like)        -   4014 e. Data is saved to TIFF and log (csv) files    -   4016. Instrument goes to step 4012 for next filter position/data        point (Repeat steps 4012 through 4016 for n filter position/data        points)    -   4018. Instrument runs initial thermal cycler conditions (e.g.        hot start)        -   4018 a. Instrument sets thermal cycler set point 1 and ramp            power (e.g. 50° C.)        -   4018 b. Instrument queries thermal cycler for “chuck”            temperature        -   4018 c. Once thermal cycler has reached set point            temperature, instrument counts down hold time for set point            1        -   4018 d. Once hold time count has elapsed, Instrument sets            thermal cycler set point 2 and ramp power (e.g. 95° C.)        -   4018 e. Once thermal cycler has reached set point            temperature, instrument counts down hold time for set point            2        -   4018 f. Once hold time count has elapsed instrument runs PCR            cycling method (Or runs further initial cycling condition            set points)    -   4020. Instrument runs n PCR cycles        -   4020 a. Instrument sets thermal cycler initial cycling set            point (e.g. annealing) and ramp power (e.g. 60° C.)        -   4020 b. Instrument queries thermal cycler for “chuck”            temperature        -   4020 c. Once thermal cycler has reached set point            temperature the instrument counts down hold time        -   4020 d. Instrument sets thermal cycler to second cycling set            point (e.g. denature/95° C.) and ramp power (e.g. 10)        -   4020 e. Instrument queries thermal cycler for “chuck”            temperature        -   4020 f. Once thermal cycler has reached set point            temperature, instrument counts down hold time for denature            set point        -   4020 g. Once hold time count has elapsed go to 4020(a)            (Instrument repeats for n cycles)    -   4022. Once n cycles completes the instrument collects the post        PCR data (Run steps 4010 through 4016. Data analysis performed        in an embodiment as described more fully throughout the present        specification.    -   4024. Once the post run data is collected the method ends (See        FIG. 38 and related description related to a generic instrument        run)    -   4026. Perform other steps as necessary.

As shown, above, the above steps can be used to perform genotyping anddata collection according to an embodiment of the present invention.Other alternatives can also be provided where steps are added, one ormore steps are removed, or one or more steps are provided in a differentsequence without departing from the scope of the claims herein. Incertain embodiments, any of these steps may be combined with the othersrecited herein.

According to embodiments of the present invention, after the genotypingmethod run is complete, and the data is collected, the data is saved andmade ready for analysis of the present or future runs. Additionally, 24hour walk away operation is provided by embodiments. Generally, thethermal cycler method run is based on current system architecture andtwo temperature PCR cycling is utilized. Alternatively, otheralternatives are added as appropriate at step 4020(g). Moreover, sampletemperature off set compensation is included in some embodiments. One ofordinary skill in the art would recognize many variations,modifications, and alternatives.

It is also understood that the examples and embodiments described hereinare for illustrative purposes only and that various modifications orchanges in light thereof will be suggested to persons skilled in the artand are to be included within the spirit and purview of this applicationand scope of the appended claims.

In an alternative embodiment of the present invention, FIG. 41A depictsa substrate 4100 of a microfluidic device that has integrated pressureaccumulator wells 4101 and 4102, each having therein a drywell 4103,4104 for receiving a valve, preferably a check valve attached to a cover(see FIG. 41B). Substrate 4100 further includes one or more well banks4106 a, b, c, and d, each having one or more wells 4105 located therein.Each of the wells 4105 of substrate 4100 have channels leading from well4105 to elastomeric block location 4107 within substrate 4100 forattaching an elastomeric block, preferably an elastomeric block formedfrom two or more layers of elastomeric material having microfabricatedrecesses or channels formed therein.

FIG. 41B depicts an exploded view of a complete microfluidic device 4199comprising the components shown in FIG. 41A, and further comprising anelastomeric block 4108 which is attached, or more preferably bonded, andyet more preferably directly bonded, preferably without use of adhesivesto the elastomeric block location 4107 of substrate 4100 to form thecomplete microfluidic device 4199 (FIG. 41C). Within elastomeric block4108 are one or more channels in fluid communication with one or morevias 4114, which in turn provide fluid communication between thechannels within the elastomeric block and channels within the substratewhich then lead to wells 4105 within well rows 4106 a-d to provide forfluid communication between wells 4105 of substrate 4100 and thechannels within elastomeric block 4108. Accumulator well tops 4109 and4110 are attached to accumulator wells 4101 and 4102 to form accumulatorchambers 4115 and 4116. Accumulator well tops 4109 and 4110 includevalves 4112 and 4111, respectively, which are preferably check valvesfor introducing and holding gas under pressure into accumulator chambers4115 and 4116. Valves 4111 and 4112 are situated inside of drywells 4102and 4104 to keep liquid, when present in accumulator chambers 4115 and4116, from contacting valves 4111 and 4112. Valves 4111 and 4112preferably may be mechanically opened by pressing a shave, pin or thelike, within a preferred check valve to overcome the self closing forceof the check valve to permit release of pressure from the accumulatorchamber to reduce the pressure of the fluid contained within theaccumulator chamber.

FIG. 41D depicts a plan view of microfluidic device 4199 and wells 4105,wherein a port is located adjacent the base of the well, preferably thebottom, or alternatively the side of well 4105 for passage of fluid fromthe well into a channel formed in substrate 4100, preferably on the sideof substrate 4100 opposite of well 4105. In a particularly preferredembodiment, substrate 4100 is molded with recesses therein, the recessesbeing made into channels by a sealing layer, preferably an adhesive filmor a sealing layer.

Substrate 4100 and its associated components may be fabricated frompolymers, such as polypropylene, polyethylene, polycarbonate,high-density polyethylene, polytetrafluoroethylene PTFE or Teflon (R),glass, quartz, or a metal (for example, aluminum), transparentmaterials, polysilicon, or the like. Accumulator well tops 4109 and 4110further may comprise access screws 4112 which can be removed tointroduce or remove gas or liquid from accumulator chambers 4115 and4116. Preferably, valves 4112 and 4111 can be actuated to release fluidpressure otherwise held inside of accumulator chambers 4115 and 4116.Notch 4117 is used to assist correct placement of the microfluidicdevice into other instrumentation, for example, instrumentation used tooperate or analyze the microfluidic device or reactions carried outtherein. FIG. 41D further depicts a hydration chamber 4150 surroundingelastomeric block region 4107, which can be covered with a hydrationcover 4151 to form a humidification chamber to facilitate the control ofhumidity around the elastomeric block. Humidity can be increased byadding volatile liquid, for example water, to humidity chamber 4151,preferably by wetting a blotting material or sponge. Polyvinyl alcoholmay preferably be used. Humidity control can be achieved by varying theratio of polyvinyl alcohol and water, preferably used to wet a blottingmaterial or sponge. Hydration can also be controlled by using a humiditycontrol device such as a HUMIDIPAK™ humidification package which, forexample, uses a water vapor permeable but liquid impermeable envelop tohold a salt solution having a salt concentration suitable formaintaining a desired humidity level. See U.S. Pat. No. 6,244,432 bySaari et al, which is herein incorporated by reference for all purposesincluding the specific purpose of humidity control devices and methods.Hydration cover 4150 is preferably transparent so as to not hindervisualization of events within the elastomeric block during use.Likewise, the portion of substrate 4100 beneath the elastomeric blockregion 4107 is preferably transparent, but may also be opaque orreflective.

FIG. 41E depicts a plan view of substrate 4100 with its channels formedtherein providing fluid communication between wells 4105 and elastomericblock 4108 (not shown) which is attached to substrate 4100 withinelastomeric block region 4107, through channels 4172. Accumulatorchambers 4101 and 4102 are in fluid communication with elastomeric blockregion 4107 and ultimately, elastomeric block 4108, through channels4170.

FIG. 41F depicts a bottom plan view of substrate 4100. In a particularlypreferred embodiment, recesses are formed in the bottom of substrate4100 between a first port 4190 which passes through substrate 4100 tothe opposite side where wells 4105 are formed and a second port 4192which passes through substrate 4100 in fluid communication with a via inelastomeric block 4108 (not shown).

FIG. 41G depicts a cross-sectional view of substrate 4100 withelastomeric block 4108 situated in elastomeric block region 4107 alongwith sealing layer 4181 attached to the side of substrate 4100 oppositeof elastomeric block 4108. Well 4105 is in fluid communication withelastomeric block 4108 through first port 4190, channel 4170, and secondport 4192 and into a recess of elastomeric layer 4108, which is sealedby a top surface 4197 of substrate 4100 to form a channel 4185. Sealinglayer 4181 forms channel 4170 from recesses molded or machined into abottom surface 4198 substrate 4100. Sealing layer 4181 is preferably atransparent material, for example, polystyrene, polycarbonate, orpolypropylene. In one embodiment, sealing layer 4181 is flexible such asin adhesive tape, and may be attached to substrate 4100 by bonding, suchas with adhesive or heat sealing, or mechanically attached such as bycompression. Preferably materials for sealing layer 4181 are compliantto form fluidic seals with each recess to form a fluidic channel withminimal leakage. Sealing layer 4181 may further be supported by anadditional support layer that is rigid (not shown). In anotherembodiment, sealing layer 4181 is rigid.

The microfluidic devices of the present invention may be used asstand-alone devices, or preferably, may be used as part of a system asprovided for by the present invention. FIG. 42A depicts a perspectiveview of a robotic station for actuating a microfluidic device. Anautomated pneumatic control and accumulator charging station 4200includes a receiving bay 4203 for holding a microfluidic device 4205 ofthe present invention such as the type depicted in FIGS. 41A-G. A platen4207 is adapted to contact an upper face 4209 of microfluidic device4205. Platen 4207 has therein ports that align with microfluidic device4205 to provide fluid pressure, preferably gas pressure, to wells andaccumulators within microfluidic device 4205. In one embodiment, platen4207 is urged against upper face 4221 of microfluidic device 4205 bymovement of an arm 4211, which hinges upon a pivot 4213 and is motivatedby a piston 4215 which is attached at one end to arm 4211 and at theother end to a platform 4217. Sensors along piston 4215 detect pistonmovement and relay information about piston position to a controller,preferably a controller under control of a computer (not shown)following a software script. A plate detector 4219 detects the presenceof microfluidic device 4205 inside of receiving bay 4203, and preferablycan detect proper orientation of microfluidic device 4205. This mayoccur, for example, by optically detecting the presence and orientationof microfluidic device 4205 by reflecting light off of the side ofmicrofluidic device 4205. Platen 4207 may be lowered robotically,pneumatically, electrically, or the like. In some embodiments, platen4207 is manually lowered to engage device 4205.

FIG. 42B depicts charging station 4200 with platen 4207 in the downposition urged against upper face 4221 of microfluidic device 4205,which is now covered by a shroud of platen 4207. In one embodiment,fluid lines leading to platen 4207 are located within arm 4211 and areconnected to fluid pressure supplies, preferably automatic pneumaticpressure supplies under control of a controller. The pressure suppliesprovide controlled fluid pressure to ports within a platen face (notshown) of platen 4207, to supply controlled pressurized fluid tomicrofluidic device 4205. Fine positioning of platen 4207 is achieved,at least in-part, by employing a gimbal joint 4223 where platen 4207attaches to arm 4211 so that platen 4207 may gimbal about an axisperpendicular to upper face 4221 of microfluidic device 4205.

FIGS. 42C and 42D provide side-views of charging station 4200 in both upand down positions, respectively. FIG. 42E depicts a close-up view ofplaten 4207 in a down position.

FIG. 42F depicts a cut-away side-view of platen 4207 urged against upperface 4221 of microfluidic device 4205. Platen 4207 is urged againstupper face 4221 of microfluidic device 4205 to form a fluid tight sealbetween microfluidic device 4205 and a platen face 4227, or betweenportions of device 4205 and face 4227. Platen face 4227, in oneembodiment, includes or is made of a compliant material such as aresilient elastomer, preferably chemical resistant rubber or the like.Inside platen 4207 are separate fluid pressure lines, preferably gaspressure lines, which mate with various locations on upper face 4221 ofmicrofluidic device 4205. Also shown are check valve purge actuators4233 which are actuated, preferably pneumatically, and which whenactuated, push a pin 4231 downward into check valve 4112 to open andrelieve fluid pressure, or permit the introduction of fluid throughcheck valve 4112 by overcoming its opening resistance. In oneembodiment, platen 4207 has first and second purge actuators 4233 whichengage check valves 4111 and 4112 (see FIG. 41B).

In another embodiment, chip or device 4205 is manufactured with normallyclosed containment and/or interface valves. In this embodiment,accumulators would not be necessary to hold valves shut duringincubation. Pressure would be applied to carrier or device 4205 wellregions when interface and/or containment valves are desired to beopened. For all or most other times, the valves would remain closed toseparate the various chip experiments from one another, and/or toseparate reagent and protein wells on the chip from one another.

FIG. 42G provides an extreme close-up view of purge actuator 4233 actingupon check valve 4112 located within substrate 4100 of microfluidicdevice 4205.

FIG. 42H depicts a cut-away view of platen 4207 urged against upper face4221 of microfluidic device 4205 wherein a pressure cavity 4255 isformed above well row 4106 by contacting platen face 4227 against aridge 4250 of upper face 4221. Fluid pressure, preferably gas pressure,is then applied to pressure cavity 4255 by introducing a fluid intocavity 4255 from pressure lines running down arm 4211 of chargingstation 4200. Pressure is regulated by pressure regulators associatedwith charging station 4200, preferably by electronically controlledvariable pressure regulators that can change output pressure inaccordance with signals sent by a charging station controller,preferably under computer control. Fluid pressure inside of pressurecavity 4255 in turn drives liquid within well 4105 through the channelswithin substrate 4100 and into channels and/or chambers of elastomericblock 4108 to fill channels or chambers or to actuate a deflectableportion of elastomeric block 4108, preferably a deflectable membranevalve as previously described.

In a particular embodiment, the integrated carrier 4100 and microfluidicdevice are adapted for performing desired experiments according toembodiments of the present invention by using the systems of the presentinvention. More specifically, as shown in FIG. 43A, a system 4300includes one or more receiving stations 4310 each adapted to receive acarrier 1400. In a particular embodiment, system 4300 includes four (4)receiving stations 4310, although fewer or a greater number of stations4310 are provided in alternative embodiments of the present invention.FIG. 43B depicts carrier 4100 and a device in combination disposed instation 4310 under an interface plate 4320. Interface plate 4320 isadapted to translate downward in FIG. 43B so that interface plate 4320engages the upper surface of carrier 4100 and its microfluidic device.In some embodiments, station 4310 and platen 4320 are similar to station4200 and platen 4207. Interface plate 4320 includes one or more ports4325 for coupling with regions in carrier 4100 which are adapted toreceive fluids, pressure, or the like. In some embodiments, interfaceplate 4320 includes two ports, three ports, four ports, five ports, sixports, seven ports, eight ports, nine ports, ten ports, or the like. Ina preferred embodiment, interface plate 4320 is coupled to six lines forproviding pressure to desired regions of carrier 1400, and two lines forproviding a mechanism for activating check valves 4111 and 4112.

FIG. 43C depicts various regions of interface plate 4320 according to aparticular embodiment of the present invention, similar to FIG. 43C. Inalternative embodiments interface plate 4320 includes a different numberor configuration of ports than those depicted in FIG. 43C.

As shown in FIG. 43A, system 4300 further includes a processor that, inone embodiment, is a processor associated with a laptop computer orother computing device 4330. Computing device 4330 includes memoryadapted to maintain software, scripts, and the like for performingdesired processes of the present invention. Further, computing device4330 includes a screen 4340 for depicting results of studies andanalyses of microfluidic devices. System 4300 is coupled to one or morepressure sources, such as a pressurized fluid, gas, or the like, fordelivering same to the microfluidic devices which are fluidly coupled tointerface plate(s) 4320.

A modified version of the device shown in FIG. 27 is shown in FIG. 44.The general structure bears many similarities with that depicted in FIG.27, and common elements in both figures share the same referencenumbers. The device 150 illustrated in FIG. 44 differs in that pairs ofhorizontal flow channels 104 are joined to a common inlet 124. Thisessentially enables duplicate sets of reagents to be introduced into twoadjacent flow channels with just a single injection into inlet 124. Theuse of a common inlet is further extended with respect to the verticalflow channels 102. In this particular example, each sample is introducedinto five vertical flow channels 102 with a single injection into sampleinlet 120. Thus, with this particular device, there are essentially tenreplicate reactions for each particular combination of sample andreagent. Of course, the number of replicate reactions can be varied asdesired by altering the number of vertical and/or horizontal flowchannels 102, 104 that are joined to a common inlet 120, 124.

The device shown in FIG. 44 also includes a separate control channelinlet 128 that regulates control channel 130 that can be used to governsolution flow toward outlets 132 and another control channel inlet 132that regulates control channel 134 that regulates solution flow tooutlets 136. Additionally, device 150 incorporates guard channels 138.In this particular design, the guard channels 138 are formed as part ofcontrol channels 116. As indicated supra, the guard channels 138 aresmaller than the row valves 108; consequently, the membranes of theguard channels 138 are not deflected into the underlying horizontal flowchannels 104 such that solution flow is disrupted.

What is claimed is:
 1. A database system for processing images providedin one or more well regions of a microfluidic device, each of the one ormore well regions arranged in a spatial orientation, the database systemcomprising: a multi-pixel image capturing device coupled to themicrofluidic device, the multi-pixel image capturing device beingadapted to capture a plurality of multi-pixel images from at least oneof the one or more well regions, each of the plurality of multi-pixelimages being captured concurrently in a first format; an imageprocessing device operably coupled to the multi-pixel image capturingdevice to provide the plurality of multi-pixel images to the imageprocessing device and further including a data processor programmed to:derive at least a first image and a second image from the plurality ofmulti-pixel images, the first image and the second image being in asecond format; and determine at least a first feature information fromthe first image; and determine a second feature information from thesecond image; and a database storage device coupled to the imageprocessing device, the database storage device being adapted to store atleast the first feature information and the second feature information.2. The database system of claim 1 wherein the processor is furtherprogrammed to process multi-pixel images comprising images of at leastone of a fluorescent species, a luminescent species, a colorometricspecies, or a radiometric species.
 3. The database system of claim 1wherein the one or more well regions comprise two or more well regionscoupled together by one or more valves.
 4. The database system of claim1 further comprising a second chip device comprising at least one ofmulti-well plates for microbatch crystallization, sitting drop vapordiffusion crystallization, hanging drop vapor diffusion crystallization,or capillary plates for liquid-liquid diffusion.
 5. The database systemof claim 1 wherein the data processor is further programmed to transformthe multi-pixel images in the first format into a second formatcomprising a transform domain.
 6. The database system of claim 1 whereinthe multi-pixel imaging capturing device is adapted to capture each ofthe plurality of multi-pixel images simultaneously.
 7. A system forprocessing images, the system comprising: a microfluidic devicecomprising one or more well regions arranged in a spatial orientation; amulti-pixel image capturing device optically coupled to the microfluidicdevice, the multi-pixel image capturing device being adapted toconcurrently capture a plurality of multi-pixel images in a first formatfrom at least one of the one or more well regions; an image processingdevice including a data processor and operable to receive the pluralityof multi-pixel images, the data processor being programmed to: derive atleast a first image and a second image from the plurality of multi-pixelimages, the first image and the second image being in a second format;and determine at least a first feature information from the first image;and determine a second feature information from the second image; and adatabase storage device coupled to the image processing device, thedatabase storage device being adapted to store at least the firstfeature information and the second feature information.
 8. The system ofclaim 7 wherein the data processor is further programmed to processmulti-pixel images of at least one of a fluorescent species, aluminescent species, a colorometric species, or a radiometric species.9. The system of claim 7 wherein the data processor is furtherprogrammed to transform the multi-pixel images in the first format intoa second format comprising a transform domain.
 10. The system of claim 7wherein the multi-pixel image capturing device is adapted tosimultaneously capture the multi-pixel images.